Glycopeptide remodeling using amidases

ABSTRACT

This invention provides methods for modifying glycosylation patterns of glycopeptides, including recombinantly produced glycopeptides. Also provided are glycopeptide compositions in which the glycopeptides have a homogeneous glycosylation pattern.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a U.S. National Phase Application ofPCT/US02/38440 filed Nov. 27, 2002, which claims priority from U.S.Provisional Application No. 60/334,233 filed Nov. 28, 2001, thedisclosures of which are hereby incorporate by reference in theirentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to the field of methods for remodelingglycopeptide to provide glycopeptides with novel and/or substantiallyuniform glycosylation patterns.

2. Background

A. Protein Glycosylation

The biological activity of many glycopeptides is highly dependent uponthe presence or absence of particular oligosaccharide structuresattached to the glycopeptide. Improperly glycosylated glycopeptides areimplicated in cancer, infectious diseases and inflammation (Dennis etal., BioEssays 21: 412-421 (1999)). Moreover, the glycosylation patternof a therapeutic glycopeptide can affect numerous aspects of thetherapeutic efficacy such as solubility, resistance to proteolyticattack and thermal inactivation, immunogenicity, half-life, bioactivity,and stability (see, e.g., Rotondaro et al., Mol. Biotechnol. 11: 117-128(1999); Lis et al., Eur. J. Biochem. 218: 1-27 (1993); Ono et al., Eur.J. Cancer 30A (Suppl. 3), S7-S11 (1994); and Hotchkiss et al., Thromb.Haemost. 60: 255-261 (1988)). Regulatory approval of therapeuticglycopeptides also requires that the glycosylation be homogeneous andconsistent from batch to batch.

Glycosylation is a complex post-translational modification that ishighly cell dependent. Following translation, proteins are transportedinto the endoplasmic reticulum (ER), glycosylated and sent to the Golgifor further processing. The resulting glycopeptides are subsequentlytargeted to various organelles, become membrane components, or they aresecreted into the periplasm.

During glycosylation, either N-linked or O-linked glycopeptides areformed. N-glycosylation is a highly conserved metabolic process, whichin eukaryotes is essential for viability. N-linked glycosylation is alsoimplicated in development and homeostasis; N-linked glycopeptidesconstitute the majority of cell-surface proteins and secreted proteins,which are highly regulated during growth and development (Dennis et al.,Science 236:582-585 (1987)). N-glycosylation is also believed to berelated to morphogenesis, growth, differentiation and apoptosis(Kukuruzinska et al, Biochem. Biophys. Acta. (in press) (1998)).

In eukaryotes, N-linked glycosylation occurs on the asparagine of theconsensus sequence Asn-X_(aa)-Ser/Thr, in which X_(aa) is any amino acidexcept proline (Kornfeld et al., Ann Rev Biochem 54:631-664 (1985);Kukuruzinska et al., Proc. Natl. Acad. Sci. USA 84:2145-2149 (1987);Herscovics et al., FASEB J. 7:540-550 (1993); and Orlean, SaccharomycesVol. 3 (1996)). O-linked glycosylation also takes place at serine orthreonine residues (Tanner et al., Biochim. Biophys. Acta. 906:81-91(1987); and Hounsell et al., Glycoconj. J. 13:19-26 (1996)). Otherglycosylation patterns are formed by linkingglycosylphosphatidylinositol to the carboxyl-terminal carboxyl group ofthe protein (Takeda et al., Trends Biochem. Sci. 20:367-371 (1995); andUdenfriend et al., Ann. Rev. Biochem. 64:593-591 (1995).

The biosynthesis of N-linked glycopeptides is initiated with thedolichol pathway in the endoplasmic reticulum (Burda, P., et al.,Biochimica et Biophysica Acta 1426:239-257 (1999); Kornfeld et al., Ann.Rev. Biochem. 54:631-664 (1985); Kukuruzinska et al., Ann. Rev. Biochem.56:915-944 (1987); Herscovics et al., FASEB J. 7:540-550 (1993)). At theheart of the dolichol pathway is the synthesis of an oligosaccharidelinked to a polyisoprenol carrier lipid. The oligosaccharide,GlcNAc₂Man₉Glc₃, is assembled through the glycosyl-transferasecatalyzed, stepwise addition of monosaccharides. The dolichol pathway ishighly conserved between yeast and mammals.

After the assembly of the dolichol-oligosaccharide conjugate, theoligosaccharide is transferred from this conjugate to an asparagineresidue of the protein consensus sequence. The transfer of theoligosaccharide is catalyzed by the multi-subunit enzymeoligosaccharyltransferase (Karaoglu et al., Cold Spring Harbor Symposiaon Quantitative Biology LX:83-92 (1995b); and Silberstein et al., FASEBJ. 10:849-858 (1996). Subsequent to the transfer of the oligosaccharideto the protein, a series of reactions, which shorten the oligosaccharideoccur. The reactions are catalyzed by glucosidases I and II andα-mannosidase (Kilker et al., J. Biol. Chem., 256:5299-5303 (1981);Saunier et al., J. Biol. Chem. 257:14155-14161 (1982); and Byrd et al.,J. Biol. Chem. 257:14657-14666 (1982)).

Following the synthesis and processing of the N-linked glycopeptide inthe endoplasmic reticulum, the glycopeptide is transported to the Golgi,where various processing steps result in the formation of the matureN-linked oligosaccharide structures. Although the dolichol pathway ishighly conserved in eukaryotes, the mature N-linked glycopeptidesproduced in the Golgi exhibit significant structural variation acrossthe species. For example, yeast glycopeptides include oligosaccharidestructures that consist of a high mannose core of 9-13 mannose residues,or extended branched mannan outer chains consisting of up to 200residues (Ballou, et al., Dev. Biol. 166:363-379 (1992); Trimble et al.,Glycobiology 2:57-75 (1992). In higher eukaryotes, the N-linkedoligosaccharides are typically high mannose, complex and mixed types ofstructures that vary significantly from those produced in yeast(Kornfeld et al., Ann. Rev. Biochem. 54:631-664 (1985)). Moreover, inyeast, a single α-1,2-mannose is removed from the central arm of theoligosaccharide, in higher eukaryotes, the removal of mannose involvesthe action of several mannosidases to generate a GlcNAc₂Man₅ structure(Kukuruzinska et al., Crit Rev Oral Biol Med. 9(4):415-448 (1998)). Thebranching of complex oligosaccharides occurs after the trimming of theoligosaccharide to the GlcNAc₂Man₅ structure. Branched structures, e.g.bi-, tri- and tetra-antennary, are synthesized by the GlcNActransferase-catalyzed addition of GlcNAc to regions of theoligosaccharide residue. Subsequent to their formation, the antennarystructures are terminated with different sugars including Gal, GalNAc,GlcNAc, Fuc and sialic acid residues.

Similar to N-glycosylation, O-glycosylation is also markedly differentbetween mammals and yeast. At the initiation of O-glycosylation,mammalian cells add a GalNAc residue directly to Ser or Thr usingUDP-GalNAc as a glycosyl donor. The saccharide unit is elongated byadding Gal, GlcNAc, Fuc and NeuNAc. In contrast to mammalian cells,lower eukaryotes, e.g., yeast and other fungi, add a mannose to Ser orThr using Man-P-dolichol as a glycosyl donor. The saccharides areelongated by adding Man and/or Gal. See, generally, Gemmill et al.,Biochim. Biophys Acta 1426: 227-237 (1999).

Efforts to elucidate the biological mechanism of protein glycosylationand the glycosylation patterns of glycopeptides had been aided by anumber of analytical techniques. For example, N-linked oligosaccharidesof recombinant aspartic protease were characterized using a combinationof mass spectrometric, 2D chromatographic, chemical and enzymaticmethods (Montesino et al., Glycobiology 9: 1037-1043 (1999)). The sameworkers have also reported the characterization of oligosaccharidesenzymatically released from purified glycopeptides usingfluorescent-labeled derivatives of the released oligosaccharides incombination with fluorophore-assisted carbohydrate electrophoresis(FACE) (Montesino et al., Protein Expression and Purification 14:197-207(1998)).

Cloned endo- and exo-glycosidases are standardly used to releasemonosaccharides and N-glycans from glycopeptides. The endoglycosidasesallow the discrimination between N-linked and O-linked glycans andbetween classes of N-glycans. Methods of separating glycopeptides onseparated glycans have also become progressively more sophisticated andselective. Methods of separating mixtures of glycopeptides and cleavedglycans have also continued to improve and techniques such as high pHanion exchange chromatography (HPAEC) are routinely used for theseparation of individual oligosaccharide isomers from a complex mixtureof oligosaccharides. Recently, a large-scale organic solvent (acetone)precipitation-based method for isolating saccharides released fromglycopeptides was reported by Verostek et al. (Analyt. Biochem. 278:111-122 (2000). Many other methods of isolating and characterizingoligosaccharides released from glycopeptides are known in the art. See,generally, Fukuda et al., GLYCOBIOLOGY: A PRACTICAL APPROACH, OxfordUniversity Press, New York 1993; and E. F. Hounsell (Ed.) GLYCOPEPTIDEANALYSIS IN BIOMEDICINE, Humana Press, Totowa, N.J., 1993.

B. Synthesis of Glycopeptides

Considerable effort has been directed towards the identification andoptimization of new strategies for the preparation of saccharides andglycopeptides derived from these saccharides. Included amongst the manypromising methods are the engineering of cellular hosts that produceglycopeptides having a desired glycosylation pattern, chemicalsynthesis, enzymatic synthesis, enzymatic remodeling of formedglycopeptides and methods that are hybrids of one or more of thesetechniques.

Cell host systems have been investigated in which glycopeptides ofinterest as pharmaceutical agents can be produced in commerciallyfeasible quantities. In principle, mammalian, insect, yeast, fungal,plant or prokaryotic cell culture systems can be used for production ofmost therapeutic and other glycopeptides. In practice, however, adesired glycosylation pattern on a recombinantly produced protein isdifficult to achieve. For example, bacteria do not N-glycosylate via thedolichol pathway, and yeast make only oligomannose-type N-glycans, whichare not generally found in humans. (see, e.g., Ailor et al. Glycobiology1: 837-847 (2000)). Similarly, plant cells do not produce sialylatedoligosaccharides, a common constituent of human glycopeptides (see,generally, Liu, Trends Biotechnol 10: 114-20 (1992); and Lerouge et al.,Plant Mol. Biol. 38: 31-48 (1998)). As recently reviewed, none of theinsect cell systems presently available the production of recombinantmammalian glycopeptides will produce glycopeptides with the same glycansnormally found when they are produced in mammals. Moreover,glycosylation patterns of recombinant glycopeptides frequently differwhen they are produced under different cell culture conditions (Watsonet al. Biotechnol. Prog. 10: 39-44 (1994); and Gawlitzek et al.,Biotechnol. J. 42: 117-131 (1995)). It now appears that glycosylationpatterns of recombinant glycopeptides can vary between glycopeptidesproduced under nominally identical cell culture conditions in twodifferent bioreactors (Kunkel et al., Biotechnol. Prog. 2000:462-470(2000). Finally, in many bacterial systems, the recombinantly producedproteins are completely unglycosylated.

Heterogeneity in the glycosylation of a recombinantly producedglycopeptides arises because the cellular machinery (e.g.,glycosyltransferases and glycosidases) may vary from species to species,cell to cell, or even from individual to individual. The substratesrecognized by the various enzymes may be sufficiently different thatglycosylation may not occur at some sites or may be vastly modified fromthat of the native protein. Glycosylation of recombinant proteinsproduced in heterologous eukaryotic hosts will often differ from thenative protein. For example, yeast and insect expressed glycopeptidestypically contain high mannose structures that are not commonly seen inhumans.

An area of great interest is the design of host cells that have theglycosylation apparatus necessary to prepare properly glycosylatedrecombinant human glycopeptides. The Chinese hamster ovary (CHO) cell isa model cell system that has been particularly well studied, because CHOcells are equipped with a glycosylation machinery that is very similarto that found in the human (Jenkins et al., Nature Biotechnol. 14:975-981 (1996)). In contrast to the many similarities between theglycosylation patterns of glycopeptides from human cells and those fromCHO cells, an important distinction exists; glycopeptides produced byCHO cells carry only α-2,3-terminal sialic acid residues, whereas thoseproduced by human cells include both α-2,3- and α-2,6-terminal sialicacid residues (Lee et al., J. Biol. Chem. 264: 13848-13855 (1989)).

Efforts to remedy the deficiencies of the glycosylation of a particularhost cell have focused on engineering the cell to express one or moremissing enzymes integral to the human glycosylation pathway. Forexample, Bragonzi et al. (Biochim. Biophys. Acta 1474: 273-282 (2000))have produced a CHO cell that acts as a ‘universal host’ cell, havingboth α-2,3- and α-2,6-sialyltransferase activity. To produce theuniversal host, CHO cells were transfected with the gene encodingexpression of α-2,6-sialyltransferase. The resulting host cells thenunderwent a second stable transfection of the genes encoding otherproteins, including human interferon γ (IFN-γ). Proteins were recoveredthat were equipped with both α-2,3- and α-2,6-sialic acid residues.Moreover, in vivo pharmacokinetic data for IFN-γ demonstrate improvedpharmacokinetics of the IFN-γ produced by the universal host, ascompared to the IFN-γ secreted by regular CHO cells transfected withIFN-γ cDNA. A similar study is reported by Weikert et al. (NatureBiotechnology 17: 1116-35 U.S.C. § 112, first paragraph (1999).

In addition to preparing properly glycosylated glycopeptides byengineering the host cell to include the necessary compliment ofenzymes, efforts have been directed to the development of both de novosynthesis of glycopeptides and the in vitro enzymatic methods oftailoring the glycosylation of glycopeptides. Methods of synthesizingboth O-linked and N-linked glycopeptides have been recently reviewed(Arsequell et al., Tetrahedron: Assymetry 8: 2839 (1997); and Arsequellet al., Tetrahedron: Assymetry 10: 2839 (1997), respectively).

Two broad synthetic motifs are used to synthesize N-linkedglycopeptides: the convergent approach; and the stepwise building blockapproach. The stepwise approach generally makes use of solid-phasepeptide synthesis methodology, originating with a glycosyl asparagineintermediate. In the convergent approach, the peptide and thecarbohydrate are assembled separately and the amide linkage betweenthese two components is formed late in the synthesis. Although greatadvances have been made in recent years in both carbohydrate chemistryand the synthesis of glycopeptides, there are still substantialdifficulties associated with chemical synthesis of glycopeptides,particularly with the formation of the ubiquitous β-1,2-cis-mannosidelinkage found in mammalian oligosaccharides. Moreover, regio- andstereo-chemical obstacles must be resolved at each step of the de novosynthesis of a carbohydrate. Thus, this field of organic synthesis lagssubstantially behind the de novo synthesis of other biomolecules such asoligonucleotides and peptides.

In view of the difficulties associated with the chemical synthesis ofcarbohydrates, the use of enzymes to synthesize the carbohydrateportions of glycopeptides is a promising approach to preparingglycopeptides. Enzyme-based syntheses have the advantages ofregioselectivity and stereoselectivity. Moreover, enzymatic synthesescan be performed using unprotected substrates. Three principal classesof enzymes are used in the synthesis of carbohydrates,glycosyltransferases (e.g., sialyltransferases,oligosaccharyltransferases, N-acetylglucosaminyltransferases),glycoaminidases (e.g., PNGase F) and glycosidases. The glycosidases arefurther classified as exoglycosidases (e.g., β-mannosidase,β-glucosidase), and endoglycosidases (e.g., Endo-A, Endo-M). Each ofthese classes of enzymes has been successfully used synthetically toprepare carbohydrates. For a general review, see, Crout et al., Curr.Opin. Chem. Biol. 2:98-111 (1998) and Arsequell, supra.

Glycosyltransferases have been used to modify the oligosaccharidestructures on glycopeptides. Glycosyltransferases have been shown to bevery effective for producing specific products with good stereochemicaland regiochemical control. Glycosyltransferases have been used toprepare oligosaccharides and to modify terminal N- and O-linkedcarbohydrate structures, particularly on glycopeptides produced inmammalian cells. For example, the terminal oligosaccharides have beencompletely sialylated and/or fucosylated to provide more consistentsugar structures which improves glycopeptide pharmacodynamics and avariety of other biological properties. For example,β-1,4-galactosyltransferase was used to synthesize lactosamine, thefirst illustration of the utility of glycosyltransferases in thesynthesis of carbohydrates (see, e.g., Wong et al., J. Org. Chem. 47:5416-5418 (1982)). Moreover, numerous synthetic procedures have made useof α-sialyltransferases to transfer sialic acid fromcytidine-5′-monophospho-N-acetylneuraminic acid to the 3-OH or 6-OH ofgalactose (see, e.g., Kevin et al., Chem. Eur. J. 2: 1359-1362 (1996)).For a discussion of recent advances in glycoconjugate synthesis fortherapeutic use see, Koeller et al., Nature Biotechnology 18: 835-841(2000).

Glycosidases normally catalyze the hydrolysis of a glycosidic bond,however, under appropriate conditions they can be used to form thislinkage. Most glycosidases used for carbohydrate synthesis areexoglycosidases; the glycosyl transfer occurs at the non-reducingterminus of the substrate. The glycosidase takes up a glycosyl donor ina glycosyl-enzyme intermediate that is either intercepted by water togive the hydrolysis product, or by an acceptor, to give a new glycosideor oligosaccharide. An exemplary pathway using a exoglycoside is thesynthesis of the core trisaccharide of all N-linked glycopeptides,including the notoriously difficult β-mannoside linkage, which wasformed by the action of β-mannosidase (Singh et al., Chem. Commun.993-994 (1996)).

Fucosyltransferases have been used in synthetic pathways to transfer afucose unit from guanosine-5′-diphosphofucose to a specific hydroxyl ofa saccharide acceptor. For example, Ichikawa prepared sialyl Lewis-X bya method that involves the fucosylation of sialylated lactosamine with acloned fucosyltransferase (Ichikawa et al., J. Am. Chem. Soc. 114:9283-9298 (1992)).

Although their use is less common than that of the exoglycosidases,endoglycosidases have also been utilized to prepare carbohydrates.Methods based on the use of endoglycosidases have the advantage that anoligosaccharide, rather than a monosaccharide, is transferred.Oligosaccharide fragments have been added to substrates usingendo-β-N-acetylglucosamines such as endo-F, endo-M (Wang et al.,Tetrahedron Lett. 37: 1975-1978); and Haneda et al., Carbohydr. Res.292: 61-70 (1996)).

In addition to their use in the preparing carbohydrates, the enzymesdiscussed above have been applied to the synthesis of glycopeptides aswell. The synthesis of a homogenous glycoform of ribonuclease B has beenpublished (Witte K. et al., J. Am. Chem. Soc. 119: 2114-2118 (1997)).The high mannose core of ribonuclease B was cleaved by treating theglycopeptide with endoglycosidase H. The cleavage occurred specificallybetween the two core GlcNAc residues. The tetrasaccharide sialyl Lewis Xwas then enzymatically rebuilt on the remaining GlcNAc anchor site onthe now homogenous protein by the sequential use ofβ-1,4-galactosyltransferase, α-2,3-sialyltransferase andα-1,3-fucosyltransferase V. Each enzymatically catalyzed step proceededin excellent yield.

Methods combining both chemical and enzymatic synthetic elements arealso known. For example, Yamamoto and coworkers (Carbohydr. Res. 305:415-422 (1998)) reported the chemoenzymatic synthesis of theglycopeptide, glycosylated Peptide T, using an endoglycosidase. TheN-acetylglucosaminyl peptide was synthesized by purely chemical means.The peptide was subsequently enzymatically elaborated with theoligosaccharide of human transferrin glycopeptide. The saccharideportion was added to the peptide by treating it with anendo-β-N-acetylglucosaminidase. The resulting glycosylated peptide washighly stable and resistant to proteolysis when compared to the peptideT and N-acetylglucosaminyl peptide T.

In conjunction with the interest in the use of enzymes to form andremodel glycopeptides, there is interest in producing enzymes that areengineered to produce desired glycosylation patterns. Methods ofproducing and characterizing mutations of enzymes of use in producingglycopeptides have been reported. For example, Rao et al. (ProteinScience 8:2338-2346 (1999) have prepared mutants ofendo-β-N-acetylglucosaminidase that are defined by structural changes,which reduce substrate binding and alter the enzyme functionality.Withers et al. (U.S. Pat. No. 5,716,812) have prepared mutantglycosidase enzymes in which the normal nucleophilic amino acid withinthe active site has been changed to a non-nucleophilic amino acid. Themutated enzymes cannot hydrolyze disaccharide products, but can stillform them.

The overall structure and the structure of the active site of bothmutated and native enzymes have been characterized by x-raycrystallography. See, e.g., van Roey et al., Biochemistry 33:13989-13996 (1994); and Norris et al., Structure 2: 1049-1059 (1994).

Despite the many advantages of the enzymatic synthesis methods set forthabove, in some cases, deficiencies remain. The preparation of properlyglycosylated glycopeptides is an exemplary situation in which additionaleffort is required and effort is being directed to improving both thesynthesis of glycopeptides and methods of remodeling biologically orchemically produced glycopeptides that are not properly glycosylated. Torealize the potential of enzymatic oligosaccharide and glycopeptidesynthesis and glycopeptide remodeling, there is a need for new syntheticapproaches. Since the biological activity of many commercially importantrecombinantly and transgenically produced glycopeptides depends upon thepresence or absence of a particular glycoform, a need exists for an invitro procedure to enzymatically modify glycosylation patterns on suchglycopeptides. The present invention fulfills these and other needs.

SUMMARY OF THE INVENTION

The present invention provides methods of remodeling the N-linkedglycosylation pattern of a glycopeptide. Typically, the methods arecarried out by glycosylating a polypeptide which comprises an Asn or anAsp residue. The protein will generally be recombinantly produced andmay be first treated chemically or with an appropriate enzyme(e.g.,endoglycanase, amidase or protease) to remove existing N-linkedcarbohydrate structure. The method can also utilize one or more steps inwhich an appropriate acceptor moiety is ligated onto the peptidestructures. The methods of the invention include contacting thepolypeptide with an activated glycosyl donor molecule (e.g., a specieshaving a leaving group) under conditions suitable for linking theactivated GlcNAc residue on the glycosyl donor molecule to an Asn or Aspresidue on the polypeptide. If desired, the glycosylation pattern of thepeptide produced using the method of the invention can be furtherelaborated using glycosylation according to the methods set forthherein, or known in the art.

The mutant amidase typically includes a substitution of an amino acidresidue for an active site acidic amino acid residue. For example, whenthe amidase is PNGase-F, the substituted active site residues willtypically be Asp at position 60, Glu at position 206 or Glu at position118.

The mutant enzyme catalyzes the reaction, usually by either of twopathways. In one pathway, the synthesis step is the reverse reaction ofthe amidase hydrolysis step. In these embodiments, the glycosyl donormolecule (e.g., a desired oligo- or mono-saccharide structure) containsa leaving group and the reaction proceeds with the addition of the donormolecule to an Asp residue on the protein. In the second pathway, thereaction proceeds with addition of the glycosyl donor to Asn residues ofthe protein. In these embodiments, the glycosyl donor molecule istypically modified with a leaving group at the reducing terminus of themolecule. For example, the leaving group can be a halogen, such asfluoride. In other embodiments the leaving group is a Asn, or aAsn-peptide moiety. In yet further embodiments, the GlcNAc residue onthe glycosyl donor molecule is modified. For example, the GlcNAc residuemay comprise a 1,2 oxazoline moiety.

The particular glycosyl donor molecule used in the methods of theinvention is not a critical aspect of the invention. Any desiredcarbohydrate structure can be added to a glycopeptide using the methodsof the invention and can be controlled to some extent depending on thesubstrate specificity of the glycosidase utilized. Typically, thestructure will comprise a bi, tri, or tetra-antennary structure commonlyfound on human glycopeptides.

The acceptor glycopeptide is also not a critical aspect of theinvention. Typically, the glycopeptide will be recombinantly expressedin a prokaryotic cell (e.g., bacterial cell, such as E coli) or in aeukaryotic cell such as a mammalian, yeast, insect, fungal or plantcell. The glycopeptide can be either a full length protein or afragment. In some embodiments, the glycopeptide can be reversiblyattached to solid support, according to well known techniques.

The invention also provides glycopeptides in which the glycosylationpattern is remodeled according to the method of the invention.Typically, at least 40% of the acceptor moieties, preferably at leastabout 60% and often at least about 80% of the targeted acceptor moietieson the glycopeptide are glycosylated. In some embodiments, theglycopeptide is reversibly immobilized on a solid support, such as anaffinity chromatography medium.

The present invention also provides methods for producing glycopeptidesthat have a glycosylation pattern, which is substantially identical tothe glycosylation pattern of a known glycopeptide. The method includescontacting a peptide or glycopeptide having an acceptor for a mutantamidase of the invention with a glycosyl donor and the mutant amidase.The transfer of the glycosyl donor onto the peptide or glycopeptide isterminated upon reaching a desired level of glycosylation. Among theuses of this aspect of the invention is the duplication oftherapeutically relevant glycopeptide structures that have been approvedor are nearing approval by a regulatory agency for use in humans. Thus,although a more (or less) thoroughly glycosylated peptide might haveimproved properties, the ability to duplicate an already approvedglycopeptide structure obviates the necessity of submitting certainglycopeptides prepared by the instant method to the full regulatoryreview process, thereby providing an important economic advantage. Thiswould allow switching from a production cell line with adequateglycosylation capabilities, but limited in expression level, to aproduction cell line that has the capability of producing significantlygreater amounts of product, but yielding an inferior glycosylationpattern. The glycosylation pattern can then be modified in vitro tomatch that of the desired product. The yield of desired glycosylatedproduct may then be increased substantially for a given bioreactor size,impacting both production economics and plant capacity. The particularglycopeptide used in the methods of the invention is generally not acritical aspect of the invention. The glycopeptide may be a fragment ora full-length glycopeptide. Typically, the glycopeptide is one that hastherapeutic use such as a hormone, a growth factor, an enzyme inhibitor,a cytokine, a receptor, a IgG chimera, or a monoclonal antibody.

Also provided are methods for the large-scale production of glycosylatedglycopeptides having a substantially uniform glycosylation pattern, andlarge-scale methods for producing glycopeptides having a knownglycosylation pattern using a mutant amidase of the invention.

The invention also provides compositions comprising the glycopeptidesprepared by the methods of the invention, and methods of using thecomposition in therapy and diagnosis.

Additional objects and advantages of the present invention will beapparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary oligosaccharides that can be added to proteinsusing the methods of the invention.

FIG. 2 shows alternate catalytic pathways for methods of the invention.

DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED EMBODIMENTSDefinitions

The following abbreviations are used herein:

-   -   Ara=arabinosyl;    -   Fru=fructosyl;    -   Fuc=fucosyl;    -   Gal=galactosyl;    -   GalA=galacturonyl;    -   GalNAc=N-acetylgalactosaminyl;    -   Glc=glucosyl;    -   GlcNAc=N-acetylglucosaninyl;    -   Man=mannosyl;    -   NeuAc=N-acetylneuraminyl.    -   NeuGc=N-glycolylneuraminyl;    -   Xyl=xylosyl.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization are those well known and commonly employedin the art. Standard techniques are used for nucleic acid and peptidesynthesis. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference),which are provided throughout this document. The nomenclature usedherein and the laboratory procedures in analytical chemistry, andorganic synthetic described below are those well known and commonlyemployed in the art. Standard techniques, or modifications thereof, areused for chemical syntheses and chemical analyses.

All oligosaccharides described herein are described with the name orabbreviation for the non-reducing saccharide (i.e., Gal), followed bythe configuration of the glycosidic bond (α or β), the ring bond (1 or2), the ring position of the reducing saccharide involved in the bond(2, 3, 4, 6 or 8), and then the name or abbreviation of the reducingsaccharide (i.e., GlcNAc). Each saccharide is preferably a pyranose. Fora review of standard glycobiology nomenclature see, Essentials ofGlycobiology Varki et al. eds. CSHL Press (1999).

Oligosaccharides are considered to have a reducing end and anon-reducing end, whether or not the saccharide at the reducing end isin fact a reducing sugar. In accordance with accepted nomenclature,oligosaccharides are depicted herein with the non-reducing end on theleft and the reducing end on the right.

The term “sialic acid” refers to any member of a family of nine-carboncarboxylated sugars. The most common member of the sialic acid family isN-acetyl-neuraminic acid(2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onicacid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member ofthe family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which theN-acetyl group of NeuAc is hydroxylated. A third sialic acid familymember is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J.Biol. Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem. 265:21811-21819 (1990)). Also included are 9-substituted sialic acids suchas a 9-O-C₁-C₆ acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of thesialic acid family, see, e.g., Varki, Glycobiology 2: 25-40 (1992);Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed.(Springer-Verlag, New York (1992)). The synthesis and use of sialic acidcompounds in a sialylation procedure is disclosed in internationalapplication WO 92/16640, published Oct. 1, 1992.

As used herein, the term “mutant amidase”, refers to an amidase of thepresent invention. Exemplary mutant amidases are produced recombinantly,however, the invention also includes the use of mutant amidases producedby chemical methods of mutation and also by synthesis of all or aportion of an amidase peptide sequence. Preferred mutant amidases lack amembrane anchoring region.

The term “recombinant” when used with reference to a cell indicates thatthe cell replicates a heterologous nucleic acid, or expresses a peptideor protein encoded by a heterologous nucleic acid. Recombinant cells cancontain genes that are not found within the native (non-recombinant)form of the cell. Recombinant cells can also contain genes found in thenative form of the cell wherein the genes are modified and re-introducedinto the cell by artificial means. The term also encompasses cells thatcontain a nucleic acid endogenous to the cell that has been modifiedwithout removing the nucleic acid from the cell; such modificationsinclude those obtained by gene replacement, site-specific mutation, andrelated techniques. A “recombinant polypeptide” is one which has beenproduced by a recombinant cell.

A “heterologous sequence” or a “heterologous nucleic acid”, as usedherein, is one that originates from a source foreign to the particularhost cell, or, if from the same source, is modified from its originalform. Thus, a heterologous glycopeptide gene in a eukaryotic host cellincludes a glycopeptide gene that is endogenous to the particular hostcell that has been modified. Modification of the heterologous sequencemay occur, e.g., by treating the DNA with a restriction enzyme togenerate a DNA fragment that is capable of being operably linked to thepromoter. Techniques such as site-directed mutagenesis are also usefulfor modifying a heterologous sequence.

A “subsequence” refers to a sequence of nucleic acids or amino acidsthat comprise a part of a longer sequence of nucleic acids or aminoacids (e.g., polypeptide) respectively.

A “recombinant expression cassette” or simply an “expression cassette”is a nucleic acid construct, generated recombinantly or synthetically,with nucleic acid elements that are capable of affecting expression of astructural gene in hosts compatible with such sequences. Expressioncassettes include at least promoters and optionally, transcriptiontermination signals. Typically, the recombinant expression cassetteincludes a nucleic acid to be transcribed (e.g., a nucleic acid encodinga desired polypeptide), and a promoter. Additional factors necessary orhelpful in effecting expression may also be used as described herein.For example, an expression cassette can also include nucleotidesequences that encode a signal sequence that directs secretion of anexpressed protein from the host cell. Transcription termination signals,enhancers, and other nucleic acid sequences that influence geneexpression, can also be included in an expression cassette.

The term “altered” refers to a peptide having a glycosylation patternthat, after application of the methods of the invention, is differentfrom that observed on the peptide as originally produced, e.g.,expressed. Typically, the oligosaccharide structures on the originallyproduced peptide are first removed by a wild-type amidase orendoglycanase and then replaced by a desired structure or structuresusing the methods of the invention.

“Peptide” and “polypeptide” are used interchangeably to refer to apolymer in which the monomers are amino acids and are joined togetherthrough amide bonds, alternatively referred to as a polypeptide.Additionally, unnatural amino acids, for example, β-alanine,phenylglycine and homoarginine are also included. Amino acids that arenot gene-encoded may also be used in the present invention. Furthermore,amino acids that have been modified to include reactive groups,glycosylation sites, polymers, therapeutic moieties, biomolecules andthe like may also be used in the invention. All of the amino acids usedin the present invention may be either the D- or L-isomer. The L-isomeris generally preferred. In addition, other peptidomimetics are alsouseful in the present invention. As used herein, “peptide” and“polypeptide” refer to both glycosylated and unglycosylated peptides.Also included are peptides that are incompletely glycosylated by asystem that expresses the peptide. For a general review, see, Spatola,A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES ANDPROTEINS, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983).

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that function in amarner similar to a naturally occurring amino acid.

“Known glycosylation pattern,” refers to a glycosylation pattern of aknown glycopeptide from any source having any known level ofglycosylation.

The term “isolated” refers to a material that is substantially oressentially free from components, which are used to produce thematerial. For glycopeptides of the invention, the term “isolated” refersto material that is substantially or essentially free from components,which normally accompany the material in the mixture used to prepare theglycopeptide. “Isolated” and “pure” are used interchangeably. Typically,isolated glycopeptides of the invention have a level of puritypreferably expressed as a range. The lower end of the range of purityfor the glycopeptides is about 60%, about 70% or about 80% and the upperend of the range of purity is about 70%, about 80%, about 90% or morethan about 90%.

When the glycopeptides are more than about 90% pure, their purities arealso preferably expressed as a range. The lower end of the range ofpurity is about 90%, about 92%, about 94%, about 96% or about 98%. Theupper end of the range of purity is about 92%, about 94%, about 96%,about 98% or about 100% purity.

Purity is determined by any art-recognized method of analysis (e.g.,band intensity on a silver stained gel, polyacrylamide gelelectrophoresis, HPLC, or a similar means).

“Essentially each member of the population,” as used herein, describes acharacteristic of a population of glycopeptides of the invention inwhich a selected percentage of the glycosyl donor moieties added to thepeptide are added to multiple, identical acceptor sites on the peptide.“Essentially each member of the population” speaks to the “homogeneity”of the sites on the peptide conjugated to a modified sugar and refers toconjugates of the invention, which are at least about 80%, preferably atleast about 90% and more preferably at least about 95% homogenous.

“Homogeneity,” refers to the structural consistency across a populationof acceptor moieties to which the glycosyl donor moieties areconjugated. Thus, in a glycopeptide of the invention in which eachglycosyl donor moiety is conjugated to an acceptor site having the samestructure as the acceptor site to which every other glycosyl donor isconjugated, the glycopeptide is the to be about 100% homogeneous.Homogeneity is typically expressed as a range. The lower end of therange of homogeneity for the glycopeptides is about 60%, about 70% orabout 80% and the upper end of the range of purity is about 70%, about80%, about 90% or more than about 90%.

When the glycopeptides are more than or equal to about 90% homogeneous,their homogeneity is also preferably expressed as a range. The lower endof the range of homogeneity is about 90%, about 92%, about 94%, about96% or about 98%. The upper end of the range of purity is about 92%,about 94%, about 96%, about 98% or about 100% homogeneity. The purity ofthe glycopeptides is typically determined by one or more methods knownto those of skill in the art, e.g., liquid chromatography-massspectrometry (LC-MS), matrix assisted laser desorption mass time offlight spectrometry (MALDITOF), capillary electrophoresis, and the like.

“Substantially uniform glycoform” or a “substantially uniformglycosylation pattern,” when referring to a glycopeptide species, refersto the percentage of acceptor moieties that are glycosylated by theglycosyl donor of interest. For example, in the case of a mutantEndo-F3, a substantially unifomn fucosylation pattern exists ifsubstantially all (as defined below) of the GlcNAc-Asn moieties areglycosylated in a glycopeptide of the invention. It is understood by oneof skill in the art, that the starting material may contain glycosylatedacceptor moieties that are glycosylated with a species having the samestructure as the glycosyl donor (typically without the leaving group).Thus, the calculated percent glycosylation includes acceptor moietiesthat are glycosylated by the methods of the invention, as well as thoseacceptor moieties already glycosylated in the starting material.

The term “substantially” in the above definitions of “substantiallyuniform” generally means at least about 40%, at least about 70%, atleast about 80%, or more preferably at least about 90%, and still morepreferably at least about 95% of the acceptor moieties for a particularmutant amidase or glycosyltransferase are glycosylated.

The practice of this invention can involve the construction ofrecombinant nucleic acids and the expression of genes in transfectedhost cells. Molecular cloning techniques to achieve these ends are knownin the art. A wide variety of cloning and in vitro amplification methodssuitable for the construction of recombinant nucleic acids such asexpression vectors are well-known to persons of skill. Examples of thesetechniques and instructions sufficient to direct persons of skillthrough many cloning exercises are found in Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology volume 152 AcademicPress, Inc., San Diego, Calif. (Berger); and Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc., (1999 Supplement) (Ausubel). Suitable host cells forexpression of the recombinant polypeptides are known to those of skillin the art, and include, for example, eukaryotic cells including insect,mammalian, plant, yeast, and fungal cells.

The Methods

Glycopeptides that have modified glycosylation patterns have importantadvantages over proteins that are in a glycosylation state that is lessthan optimal for a particular application. Such non-optimalglycosylation patterns can arise when a recombinant glycopeptide isproduced in a cell that does not have the proper complement ofglycosylation machinery to produce the desired glycosylation pattern.The optimal or preferred glycosylation pattern may or may not be thenative glycosylation pattern of the glycopeptide when produced in itsnative cell.

The biological activity of many glycopeptides depends upon the presenceor absence of a particular glycoform; thus the methods of the inventionare useful for obtaining a composition of a glycopeptide that has anincreased level of a desired biological activity compared to theglycopeptide prior to application of the methods of the invention. Forexample, increased glycosylation at an acceptor moiety will render aglycopeptide highly multivalent, thereby increasing the biologicalactivity of the glycopeptide. Other advantages of glycopeptidecompositions that have desired glycosylation patterns include, forexample, increased therapeutic half-life of a glycopeptide due toreduced clearance rate. Altering the glycosylation pattern can also maskantigenic determinants on foreign proteins, thus reducing or eliminatingan immune response against the protein. Alteration of the glycoform of aglycopeptide-linked saccharide can also be used to target a protein to aparticular tissue or cell surface receptor that is specific for thedesired oligosaccharide. The desired oligosaccharide can also be used toinhibit interactions between a receptor and its natural ligand.

In contrast to known chemical and enzymatic peptide elaborationstrategies, the methods of the invention, make it possible to assemblepeptides and glycopeptides that have a substantially homogeneousderivatization pattern. The methods are also practical for large-scaleproduction of modified peptides and glycopeptides. Thus, the methods ofthe invention provide a practical means for large-scale preparation ofglycopeptides having preselected uniform derivatization patterns. Themethods are particularly well suited for modification of therapeuticpeptides, including but not limited to, glycopeptides that areincompletely glycosylated during production in cell culture cells (e.g.,mammalian cells, insect cells, plant cells, fimgal cells, yeast cells,or prokaryotic cells) or transgenic plants or animals.

In a first aspect, the invention provides a method for modifying theglycosylation pattern of a polypeptide comprising an acceptor moiety fora first mutant amidase. The method includes contacting the polypeptidewith a reaction mixture that comprises a glycosyl donor moiety and thefirst mutant amidase under appropriate conditions to transfer a glycosylresidue from the glycosyl donor moiety to the acceptor moiety, such thatthe resulting glycopeptide has a substantially uniform glycosylationpattern.

In another aspect, the present invention provides method for preparingindustrially relevant quantities of peptides having a selectedglycosylation pattern. Thus, there is provided a large-scale method formodifying the glycosylation pattern of a polypeptide that includes anacceptor moiety for a mutant amidase. The method includes contacting atleast about 500 mg of the polypeptide with a reaction mixture thatincludes a glycosyl donor moiety for the mutant amidase and the mutantamidase under conditions appropriate to transfer a glycosyl residue fromthe glycosyl donor moiety to the acceptor moiety, thereby producing theglycopeptide having the modified glycosylation pattern.

In yet a further aspect, the invention provides a large scale method forpreparing a peptide that has a glycosylation pattern that issubstantially identical to that of a known glycopeptide. In an exampleof this aspect, the method includes contacting at least about 500 mg ofa polypeptide with a reaction mixture that comprises a glycosyl donormoiety and a mutant amidase under conditions appropriate to transfer aglycosyl residue from the glycosyl donor moiety to a glycosyl acceptormoiety on the polypeptide. The reaction is allowed to proceed for apreselected period of time and is then terminated when the glycosylationpattern is substantially identical to the known glycosylation pattern isobtained.

The invention provides compositions that include glycopeptide speciesthat have a substantially uniform N-linked glycosylation pattern.Methods and kits for obtaining such compositions are also provided. Themethods of the invention are useful for remodeling or altering theglycosylation pattern present on a peptide or glycopeptide upon itsinitial expression.

The methods of the invention provide compositions of glycopeptides thathave a substantially uniform glycosylation pattern. The methods are alsopractical for large-scale production of modified glycopeptides. Thus,the methods of the invention provide a practical means for large-scalepreparation of glycopeptides having desired glycosylation patterns. Themethods are well suited for modification of therapeutic glycopeptidesthat are incompletely glycosylated during production in cell culturecells (e.g., mammalian cells, insect cells, plant cells, fungal cells,yeast cells, or prokaryotic cells) or transgenic plants or animals.Moreover, the methods are of general utility for converting a non-humanglycoform to a human glycoforn. Further, the methods can be used toconjugate a carbohydrate having a particular property (e.g., tissuetargeting, enhancing in vivo residence, etc.) onto a peptide. Theprocesses provide an increased and consistent level of a desiredN-linked glycoform on glycopeptides present in a composition.

In an exemplary embodiment, the method of the invention further includescontacting a polypeptide with a glycosyltransferase in addition to amutant amidase. For example, in one embodiment, the polypeptidecomprises an acceptor moiety for a glycosyltransferase. The methodfurther includes contacting the polypeptide with a reaction mixture thatcomprises a glycosyl donor moiety and the glycosyltransferase underappropriate conditions to transfer a glycosyl residue from the glycosyldonor moiety to the acceptor moiety. In a preferred embodiment, theresulting polypeptide has a substantially uniform glycosylation pattern.In yet another preferred embodiment, the glycosyltransferase is selectedfrom fucosyltransferases, sialyltransferases and combination thereof Inthose embodiments in which one or more glycosyltransferase is utilizedin addition to the mutant amidase, the precursor peptide or glycopeptidemay be contacted with one or more glycosyltransferases substantiallysimultaneously. Alternatively, the precursor peptide or glycopeptide iscontacted with one or more glycosyltransferase and the mutant amidasesubstantially simultaneously. The method of the invention optionallyconsists of two or more individual steps utilizing one or more enzyme.

The methods of the invention are practiced successfully withsubstantially any peptide or glycopeptide. When the peptide orglycopeptide does not include an appropriate acceptor moiety, it iswithin the scope of the present invention to add the appropriate moietyby enzymatic and/or chemical methods. The methods of the inventiongenerally provide a pure, homogeneous glycopeptide that is characterizedby a substantially uniform glycosylation pattern.

The acceptor peptide (glycosylated or non-glycosylated) is typicallysynthesized de novo, or recombinantly expressed in a prokaryotic cell(e.g., bacterial cell, such as E. coli) or in a eukaryotic cell such asa mammalian, yeast, insect, fungal or plant cell. The peptide can beeither a full-length protein or a fragment.

The method of the invention also provides for modification ofincompletely glycosylated peptides that are produced recombinantly. Manyrecombinantly produced glycopeptides are incompletely glycosylated,exposing carbohydrate residues that may have undesirable properties,e.g., immunogenicity, recognition by the RES. Exemplary peptides thatcan be modified using the methods of the invention are set forth inTable 1.

TABLE 1 Hormones and Growth Factors G-CSF GM-CSF TPO EPO EPO variantsNESP alpha-TNF Leptin Enzymes and Inhibitors t-PA t-PA variantsUrokinase Factors VII, VIII, IX, X Dnase GlucocerebrosidaseAlpha-glucosidase iduronidase Hirudin α1 antitrypsin Antithrombin IIICytokines and Chimeric Cytokines Interleukin-1 (IL-1), 1B, 2, 3, 4Interferon-alpha (IFN-alpha) IFN-alpha-2a or b IFN-beta IFN-gammaIFN-omega Chimeric diptheria toxin-IL-2 Receptors and Chimeric ReceptorsCD4 Tumor Necrosis Factor (TNF) receptor Alpha-CD20 MAb-CD20MAb-alpha-CD3 MAb-TNF receptor MAb-CD4 PSGL-1 MAb-PSGL-1 ComplementGlyCAM or its chimera N-CAM or its chimera Monoclonal Antibodies(Immunoglobulins) MAb-anti-RSV MAb-anti-IL-2 receptor MAb-anti-CEAMAb-anti-platelet IIb/IIIa receptor MAb-anti-EGF MAb-anti-Her-2 receptorRemicade Cells Red blood cells White blood cells (e.g., T cells, Bcells, dendritic cells, macrophages, NK cells, neutrophils, monocytesand the like Stem cells

Other exemplary peptides that are modified by the methods of theinvention include members of the immunoglobulin family (e.g.,antibodies, MHC molecules, T cell receptors, and the like),intercellular receptors (e.g., integrins, receptors for hormones orgrowth factors and the like) lectins, and cytokines (e.g.,interleukins). Additional examples include tissue-type plasminogenactivator (t-PA), renin, clotting factors such as factor VIII and factorIX, bombesin, thrombin, hematopoietic growth factor, colony stimulatingfactors, viral antigens, complement proteins, α1-antitrypsin,erythropoietin, P-selectin glycopeptide ligand-1 (PSGL-1),granulocyte-macrophage colony stimulating factor, anti-thrombin III,interleukins, interferons, proteins A and C, fibrinogen, herceptin,leptin, glycosidases, among many others. This list of polypeptides isexemplary, not exclusive. The methods are also useful for modifyingchimeric proteins, including, but not limited to, chimeric proteins thatinclude a moiety derived from an immunoglobulin, such as IgG. Stillfurther exemplary peptides, which can be modified by the methods of theinvention are set forth in Appendix 1.

Peptides modified by the methods of the invention can be synthetic orwild-type peptides or they can be mutated peptides, produced by methodsknown in the art, such as site-directed mutagenesis. Glycosylation ofpeptides is typically either N-linked or O-linked. An exemplaryN-linkage is the attachment of the modified sugar to the side chain ofan asparagine residue. The tripeptide sequences asparagine-X-serine andasparagine-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of a carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a polypeptide creates a potentialglycosylation site. O-linked glycosylation refers to the attachment ofone sugar (e.g., N-acetylgalactosamine, galactose, mannose, GlcNAc,glucose, fucose or xylose) to the hydroxy side chain of a hydroxyaminoacid, preferably serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Moreover, in addition to peptides, the methods of the present inventioncan be practiced with other biological structures (e.g., whole cells,and the like).

In certain embodiments, a glycosylation site not present in the wildtype peptide is added to the peptide upon which the method of theinvention is practiced. Addition of glycosylation sites to a peptide isconveniently accomplished by altering the amino acid sequence such thatit contains one or more glycosylation sites. The addition may also bemade by the incorporation of one or more species presenting an carboxylor carboxyamide group, preferably aspartic acid or asparagine, withinthe sequence of the peptide (for N-linked glycosylation site). For ease,the peptide amino acid sequence is preferably altered through changes atthe DNA level, particularly by mutating the DNA encoding the peptide atpreselected bases such that codons are generated that will translateinto the desired amino acids. The DNA mutation (s) are preferably madeusing methods known in the art.

Addition or removal of any carbohydrate moieties present on the peptideor glycopeptide is accomplished either chemically or enzymatically.Chemical deglycosylation is preferably brought about by exposure of thepolypeptide to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the peptide intact. Chemicaldeglycosylation is described by Hakimuddin et al., Arch. Biochem.Biophys. 259: 52 (1987) and by Edge et al., Anal. Biochem. 118: 131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptidevariants can be achieved by the use of a variety of amidases and endo-and exo-glycosidases as described by Thotakura et al., Meth. Enzymol.138: 350 (1987).

Chemical addition of glycosyl moieties is carried out by anyart-recognized method. Enzymatic addition of sugar moieties ispreferably achieved using a modification of the methods set forthherein, substituting native glycosyl units for the modified sugars usedin the invention. Other methods of adding sugar moieties are disclosedin U.S. Pat. Nos. 5,876,980, 6,030,815, 5,728,554, and 5,922,577.

Exemplary attachment points for selected glycosyl residues include, butare not limited to: (a) consensus sites for N- and O-glycosylation; (b)terminal glycosyl moieties that are acceptors for a glycosyltransferase;(c) arginine, asparagine and histidine; (d) free carboxyl groups; (e)free sulfhydryl groups such as those of cysteine; (f) free hydroxylgroups such as those of serine, threonine, or hydroxyproline; (g)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan; or (h) the amide group of glutamine. Exemplary methods ofuse in the present invention are described in WO 87/05330 published Sep.11, 1987, and in Aplin and Wriston, CRC CRIT. REV. BIOCHEM., pp. 259-306(1981).

The glycosylation pattern of immunoglobulins, as well as chimericproteins that include all or part of an immunoglobulin, such as animmunoglobulin heavy chain constant region, also affects biologicalactivity. Oligosaccharides attached to IgG molecules purified from humansera, in particular the oligosaccharides attached to Asn297 of IgG, areimportant for IgG structure and function (Rademacher and Dwek (1983).Prog. Immunol 5: 95-112; Jefferies et al. (1990)). The absence of theseoligosaccharides results in a lack of binding to the monocyte Fcreceptor, a decline in complement activation, an increase insusceptibility to proteolytic degradation, and reduced clearance fromcirculation of antibody-antigen complexes. Immunoglobulinoligosaccharides, in particular those of IgG, naturally exhibit highmicroheterogeneity in their structures (Kobata (1990) Glycobiology 1:5-8). Therefore, use of the methods of the invention to provide a moreuniform glycopeptide results in an improvement of one or more of thesebiological activities (e.g. enhanced complement activation, increasedbinding to the monocyte Fc receptor, reduced proteolysis, and increasedclearance of antibody-antigen complexes). The methods of the inventionare also useful for modifying oligosaccharides on other immunoglobulinsto enhance one or more biological activities. For example, high-mannoseoligosaccharides are generally attached to IgM and IgD. Sucholigosaccharides can be modified as described herein to yield antibodieswith enhanced properties.

Peptides modified by the methods of the invention can be synthetic orwild-type peptides or they can be mutated peptides, produced by methodsknown in the art, such as site-directed mutagenesis. Glycosylation ofpeptides is typically either N-linked or O-linked. An exemplaryN-linkage is the attachment of the modified sugar to the side chain ofan asparagine residue. The tripeptide sequences asparagine-X-serine andasparagine-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of a carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a polypeptide creates a potentialglycosylation site. As will be apparent to those of skill in the art, inthis method, asparagine is optionally replaced by aspartic acid.O-linked glycosylation refers to the attachment of one sugar (e.g.,N-acetylgalactosamine, galactose, mannose, GlcNAc, glucose, fucose orxylose) to a the hydroxy side chain of a hydroxyamino acid, preferablyserine or threonine, although 5-hydroxyproline or 5-hydroxylysine mayalso be used.

Moreover, in addition to peptides, the methods of the present inventioncan be practiced with other biological structures (e.g., whole cells,and the like).

The present invention is also useful in conjunction with methods thatgraft a glycosylation site onto a peptide at a location that does nothave such a site upon expression. Addition of glycosylation sites to apeptide is conveniently accomplished by altering the amino acid sequencesuch that it contains one or more glycosylation sites. The addition mayalso be made by the incorporation of one or more species presenting an—NH₂ group, preferably Arg or Asn residues, within the sequence of thepeptide (for NH₂-linked glycosylation sites). An Asp can also be used.For ease, the peptide amino acid sequence is preferably altered throughchanges at the DNA level, particularly by mutating the DNA encoding thepeptide at preselected bases such that codons are generated that willtranslate into the desired amino acids. The DNA mutation (s) arepreferably made using methods known in the art.

The present invention also provides means of adding one or more selectedglycosyl residues to a peptide, either before or after the amidase hasconjugated a carbohydrate to at least one of the selected amino acidresidues of the peptide. The present embodiment is useful, for example,when it is desired to conjugate a carbohydrate moiety to a selectedglycosyl residue that is either not present on a peptide or is notpresent on the peptide in a desired amount. Thus, prior to coupling adonor carbohydrate moiety to a peptide, the acceptor glycosyl residue isconjugated to the peptide by enzymatic or chemical coupling. In anotherembodiment, the glycosylation pattern of a glycopeptide is altered priorto the conjugation of the donor carbohydrate moiety by the removal of acarbohydrate residue from the glycopeptide to form a desired acceptormoiety. See, for example WO 98/31826.

Addition or removal of any carbohydrate moieties present on theglycopeptide is accomplished either chemically or enzymatically.Chemical deglycosylation is preferably brought about by exposure of thepolypeptide variant to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the peptide intact. Chemicaldeglycosylation is described by Hakimuddin et al., Arch. Biochem.Biophys. 259: 52 (1987) and by Edge et al., Anal. Biochem. 118: 131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptidevariants can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol. 138:350 (1987).

Chemical addition of glycosyl moieties is carried out by anyart-recognized method. Enzymatic addition of sugar moieties ispreferably achieved. See, e.g., U.S. Pat. Nos. 5,876,980, 6,030,815,5,728,554, and 5,922,577.

Exemplary attachment points for selected glycosyl residue include, butare not limited to: (a) consensus sites for N- and O-glycosylation; (b)terminal glycosyl moieties that are acceptors for a glycosyltransferase;(c) arginine, asparagine and histidine; (d) free carboxyl groups; (e)free sulfhydryl groups such as those of cysteine; (f) free hydroxylgroups such as those of serine, threonine, or hydroxyproline; (g)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan; or (h) the amide group of glutamine. Exemplary methods ofuse in the present invention are described in WO 87/05330 published Sep.11, 1987, and in Aplin and Wriston, CRC CRIT. REV. BIOCHEM., pp. 259-306(1981).

The methods of the invention use mutant amidases to add glycosidiclinkages to asparagine or aspartic acid residues on glycopeptides. Themutant amidases are derived from amidases capable of cleaving the C—Nbond of the glycosylated asparagine side chain, converting theasparagine to aspartic acid and liberating ammonia and the attachedglycan. Examples of amidases useful in the invention includepeptide-N₄-(N-acetyl-β-glucosaminyl)asparagine amidases (EC 3.5.1.52).Example of this class of enzyme include peptide:N-glycosidase F (PNGaseF) derived from Flavobacterium meningosepticum (Tarentino et al. J.Biol. Chem. 265:6961-6966 (1990);APADNTVNIKTFDKVKNAFGDGLSQSAEGTFTFPADVTTVKTIKMFIKNECPNKTCDEWDRYANVYVKNKTTGEWYEIGRFITPYWVGTEKLPRGLEIDVTDFKSLLSGNTELKIYETWLAKGREYSVDFDIVYGTPDYKYSAVVPVIQYNKSSIDGVPYGKAHTLGLKKNIQLPTNTEKAYLRTTISGWGHAKPYDAGSRGCAEWCFRTHTIAINNANTFQHQLGALGCSANPINNQSPGNWAPDRAGWCPGMAVPTRIDVLNNSLTGSTFSYEYKFQSWTNNGTNGDAFYAISSFVIAKSNTPISAPVVTN (SEQ ID NO:01)), and almondemulsin peptide:N-glycosidase (PNGase-A). (Plummer et al. J. Biol. Chem.256 (1981) 10243-10246 (1981)). Usually, the peptide linkage that isrecognized by these enzymes includes Sugar-Asn-X-Ser (Thr)- or Asp-X-Ser(Thr) which is the normal peptide consensus sequence recognized byenzymes that introduce N-linked sugars through a cells normalbiosynthetic pathway.

Other enzymes with similar activity that can also be used in the presentinvention include glycosylasparaginases(N4)-α-N-acetylglucosaminyl)-L-asparaginases, EC 3.5.1.26) such asmammalian or plant lysosomal glycosylasparaginases or bacterialglycosylaspariginases (see, Tollersrud et al. Biochem. 282:891-897(1992) and Tarentino et al. Biochem. Biophys. Res. Commun. 197:179-186(1993)).

The present invention is based on the observation that amidases such asthose described above can be converted from a degradative enzyme to asynthetic enzyme. The change in the catalytic activity is induced bymodifying amino acid residues of the enzyme to facilitate thisconversion. Thus modified, the enzyme is able to add more product to theglycopeptide than it cleaves. Point mutations as well as entire peptidesubstitutions can be used to improve the synthetic capabilities of theenzyme. Typically these enzymes have two or more carboxylic acid groupsin the active site of the enzyme. The present invention provides mutantforms of the enzymes noted above in which one or more of the carboxylicacid amino acids in the active site have been replaced with a differentamino acid. Such mutations provide enzymes which do not catalyze thehydrolysis of oligosaccharides, but which nevertheless retain activityto synthesize oligosaccharides with good control over thestereochemistry and regiochemistry of the reaction.

Thus, in general, the substitution will involve replacing a glutamicacid or aspartic acid residue of the wild-type enzyme with alanine,glycine, valine, leucine, isoleucine, serine, threonine, cysteine,methionine, asparagine, glutamine, histidine, proline, phenylalanine, ortyrosine. Preferably, the substituted amino acid will have a side chainof approximately equal or smaller size to the side chain of thewild-type amino acid residue to avoid significant changes to the sizeand shape of the active site. Enzymes mutated in this way are inactivewith the normal substrates, and thus cannot hydrolyze oligosaccharideproducts. They can, however, catalyze the coupling of modified glycosyldonor molecules to modified acceptors.

There are many ways known to those skilled in the art to mutate apeptide-N⁴-β-N-acetylglucosamine asparagine peptidase F (PNGaseF),PNGaseAt, PNGaseA, or glycosylasparaginase to generate an enzyme capableof catalyzing the reaction shown in FIG. 2. For example, aFlavobacterium meningosepticum PNGaseF gene is synthesized and codonoptimized for expression in E. coli. In addition, this synthetic gene isdesigned to preserve the peptide sequence but also to introduceconvenient and unique restriction endonuclease sites on either side ofthe catalytic site residues (Asp60, Glu206 and Glu118). In additionunique restriction sites would be engineered around the important aminoacid residues Trp120, Arg248 and His193. PCR primers that introduce Ser,Gly, Ala, Gln or Asn at one or more of these amino acids are designedthat also encode the unique restriction endonuclease sites on the 5′ and3′ sides of these amino acid residues. The PCR product containing themutated amino acid (s) is then subcloned into an appropriate inducibleexpression vector that allows expression of the mutated PNGaseF gene inE. coli. The mutated PNGaseF is then assayed for its ability to catalyzethe reaction shown in FIG. 2.

The site for mutation in the particular enzyme can be identified usingstandard techniques. For example, the site can be identified aftertrapping of the glycosyl-enzyme intermediate in the active site. Theintermediate may be trapped, for example, by rapid denaturation of theenzyme after contact with the substrate. Alternatively, the intermediatemay be trapped using a modified substrate which forms a relativelystable glycosyl-enzyme intermediate. Once this intermediate has beentrapped, the labeled enzyme is then cleaved into peptides by use of aprotease or by specific chemical degradation, and the peptide bearingthe sugar label then located in a chromatogram or other separationmethod and its amino acid sequence determined. Comparison of thissequence with that of the intact enzyme readily identifies the aminoacid of interest.

The catalytic residues may also be identified in the three-dimensionalstructure of the enzyme determined by X-ray crystallography or NMRspectroscopy by inspection of the active site region, searching forlikely active site residues, e.g., a Glu or Asp residue. For example,using analysis of the crystal structure and site directed mutagenesis ofPNGase F, the active site has been characterized, including the sugarbinding and catalytic sites (see, Norris et al. Structure 2:1049 (1994)and Kuhn et al. J. Biol. Chem. 270:29493-29497 (1995)).

In addition to modification of the catalytic residues, alterations ofthe sugar binding site can also be made to change the specificity of theenzyme for the oligosaccharides portion of the substrate. Pointmutations or protein reengineering can be used to change this enzymessugar specificity, according to standard techniques.

Once the active site residues are identified in one enzyme, thehomologous residues in related enzymes can also be found using standardsequence comparison programs. Methods of alignment of sequences forcomparison are well-known in the art. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection.

Mutant genes are typically prepared using site directed mutagenesis toarrive at the desired result. Methods for introducing mutations intopolynucleotide sequences are well known. Such well-known methods includesite-directed mutagenesis, PCR amplification using degenerateoligonucleotides, and other well-known techniques. See, e.g., Gilimanand Smith (1979) Gene 8:81-97, Roberts et al. (1987) Nature 328:731-734.

Mutant enzymes according to the invention may be purified from thegrowth medium of the host organism by column chromatography, for exampleon DEAE-cellulose if desired. High levels of purity are not required foruse in catalyzing oligosaccharide synthesis, however, provided thatimpurities with wild-type glycosidase activity must be substantiallyabsent.

The mutant enzymes of the invention are used to couple modified glycosyldonors with glycoside acceptors. Any desired carbohydrate structure canbe added to a peptide using the methods of the invention. Typically, thestructure will be a monosaccharide, but the present invention is notlimited to the use of modified monosaccharide sugars; oligosaccharidesand polysaccharides are useful as well.

In other embodiments, the glycosyl donor is an activated sugar.Activated sugars, which are useful in the present invention aretypically glycosides which have been synthetically altered to include anleaving group. As used herein, the term “leaving group” refers to thosemoieties, which are easily displaced in enzyme-regulated nucleophilicsubstitution reactions. Many activated sugars are known in the art. See,for example, Vocadlo et al., In CARBOHYDRATE CHEMISTRY AND BIOLOGY, Vol.2, Ernst et al. Ed., Wiley-VCH Verlag: Weinheim, Germany, 2000; Kodamaet al., Tetrahedron Lett. 34: 6419 (1993); Lougheed, et al., J. Biol.Chem. 274: 37717 (1999)).

Examples of activating groups include fluoro, chloro, bromo, tosylateester, mesylate ester, triflate ester and the like. Preferred activatedleaving groups, for use in the present invention, are those that do notsignificantly sterically encumber the enzymatic transfer of theglycoside to the acceptor. Accordingly, preferred embodiments ofactivated glycoside derivatives include glycosyl fluorides and glycosylmesylates, with glycosyl fluorides being particularly preferred. Amongthe glycosyl fluorides, α-galactosyl fluoride, α-mannosyl fluoride,α-glucosyl fluoride, α-fucosyl fluoride, α-xylosyl fluoride, α-sialylfluoride, α-N-acetylglucosaminyl fluoride, α-N-acetylgalactosaminylfluoride, β-galactosyl fluoride, β-mannosyl fluoride, β-glucosylfluoride, β-fucosyl fluoride, β-xylosyl fluoride, β-sialyl fluoride,β-N-acetylglucosaminyl fluoride and β-N-acetylgalactosaminyl fluorideare most preferred.

By way of illustration, glycosyl fluorides can be prepared from the freesugar by first acetylating the sugar and then treating it withHF/pyridine. This generates the thermodynamically most stable anomer ofthe protected (acetylated) glycosyl fluoride (i.e., the α-glycosylfluoride). If the less stable anomer (i.e., the β-glycosyl fluoride) isdesired, it can be prepared by converting the peracetylated sugar withHBr/HOAc or with HCl to generate the anomeric bromide or chloride. Thisintermediate is reacted with a fluoride salt such as silver fluoride togenerate the glycosyl fluoride. Acetylated glycosyl fluorides may bedeprotected by reaction with mild (catalytic) base in methanol (e.g.NaOMe/MeOH). In addition, many glycosyl fluorides are commerciallyavailable.

The donor molecules can be prepared according to standard techniques.For example, glycosyl fluorides can be prepared as generally describedin U.S. Pat. No. 5,716,812 or through an imidate intermediate asdescribed by Dullenkopf et al. Carbohydr Res 296:135-47 (1996). Otheractivated glycosyl derivatives can be prepared using conventionalmethods known to those of skill in the art. For example, glycosylmesylates can be prepared by treatment of the fully benzylatedhemiacetal form of the sugar with mesyl chloride, followed by catalytichydrogenation to remove the benzyl groups.

Preferred donor molecules are halogenated compounds such as glycosylfluorides or glycosyl chlorides, although other groups which arereasonably small and which function as relatively good leaving groupscan also be used. Examples of other glycosyl donor molecules includeglycosyl-Asn, glycosyl-Asn-peptide, glycosyl chlorides, glycosylacetates, glycosyl propionates, and glycosyl pivaloates, and glycosylmolecules modified with substituted phenols.

An exemplary donor molecule of the invention includes a glycosyl residueof the following formula.

In Formula 1, the symbol R represents substituted or unsubstituted alkylor aryl.

The particular saccharides coupled to the protein are not a criticalaspect of the invention. Typically, the oligosaccharides will includeany bi-, tri- and tetra-antennary structures of N-linked structures.High mannose and hybrid structures can also be transferred includingthose containing mannose-6-phophate. FIG. 1 provides a summary ofexemplary structures that can used in the invention.

In addition to the mutant amidase, the oligosaccharide structures on apeptide can be modified using a single glycosyltransferase or acombination of glycosyltransferases. For example, one can use acombination of a sialyltransferase and a galactosyltransferase. In thoseembodiments using more than one enzyme, the enzymes and substrates arepreferably combined in an initial reaction mixture, or the enzymes andreagents for a second enzymatic reaction are added to the reactionmedium once the first enzymatic reaction is complete or nearly complete.By conducting two enzymatic reactions in sequence in a single vessel,overall yields are improved over procedures in which an intermediatespecies is isolated. Moreover, cleanup and disposal of extra solventsand by-products is reduced.

In a preferred embodiment, each of the first and second enzyme is aglycosyltransferase. In another preferred embodiment, one enzyme is anendoglycosidase. In an additional preferred embodiment, more than twoenzymes are used to assemble the modified glycopeptide of the invention.The enzymes are used to alter a saccharide structure on the peptide atany point either before or after the addition of the modified sugar tothe peptide.

In another preferred embodiment, each of the enzymes utilized to producea conjugate of the invention are present in a catalytic amount. Thecatalytic amount of a particular enzyme varies according to theconcentration of that enzyme's substrate as well as to reactionconditions such as temperature, time and pH value. Means for determiningthe catalytic amount for a given enzyme under preselected substrateconcentrations and reaction conditions are well known to those of skillin the art.

The temperature at which an above process is carried out can range fromjust above freezing to the temperature at which the most sensitiveenzyme denatures. Preferred temperature ranges are about 0° C. to about45° C., and more preferably about 20° C. to about 30° C. In anotherexemplary embodiment, one or more components of the present method areconducted at an elevated temperature using a thermophilic enzyme.

The reaction mixture is maintained for a period of time sufficient forthe acceptor to be glycosylated, thereby forming the desired conjugate.Some of the conjugate can often be detected after a few hours, withrecoverable amounts usually being obtained within 24 hours or less.Those of skill in the art understand that the rate of reaction isdependent on a number of variable factors (e.g, enzyme concentration,donor concentration, acceptor concentration, temperature, solventvolume), which are optimized for a selected system.

The present invention also provides for the industrial-scale productionof modified peptides. On an industrial scale, it may be advantageous toimmobilize the amidase on a solid support to facilitate its removal froma batch of product and subsequent reuse. Such immobilization can beaccomplished by use of a fusion protein in which the mutant glycoside isengineered onto another protein with high affinity for an insolublematrix. Techniques for immobilizing proteins on solid supports are wellknown in the art. For example, a fusion protein with a cellulose bindingprotein prepared in the manner described by Ong et al., Biotechnology7:604-607 (1989) could be used in accordance with the invention.

In other embodiments, the target glycopeptide is immobilized on a solidsupport. Preferably, the target glycopeptide is reversibly immobilizedso that the glycopeptide can be released after the glycosylationreaction is completed. The term “solid support” also encompassessemi-solid supports. Many suitable matrices are known to those of skillin the art. Ion exchange, for example, can be employed to temporarilyimmobilize a glycopeptide on an appropriate resin while theglycosylation reaction proceeds. A ligand that specifically binds to theglycopeptide of interest can also be used for affinity-basedimmobilization. Antibodies that bind to a glycopeptide of interest aresuitable; where the glycopeptide of interest is itself an antibody orfragment thereof, one can use protein A or G as the affinity resin. Dyesand other molecules that specifically bind to a protein of interest thatis to be glycosylated are also suitable.

In the discussion that follows, methods of use in conjunction with theinvention are exemplified by the conjugation of sialic acid moiety to apeptide, which is glycosylated by a method of the invention. The focusof the following discussion on the use of sialic acid and glycosylatedpeptides is for clarity of illustration and is not intended to implythat the invention is limited to the conjugation of these two partners.One of skill understands that the discussion is generally applicable tothe additions of glycosyl moieties other than sialic acid.

In general, an acceptor for the sialyltransferase is present on thepeptide to be modified by the methods of the present invention either asa naturally occurring structure or one placed there recombinantly,enzymatically or chemically. Suitable acceptors, include, for example,galactosyl acceptors such as Galβ1,4GlcNAc, Galβ1,4GalNAc,Galβ1,3GalNAc, lacto-N-tetraose, Galβ1,3GlcNAc, Galβ1,3Ara,Galβ1,6GlcNAc, Galβ1,4Glc (lactose), and other acceptors known to thoseof skill in the art (see, e.g., Paulson et al., J. Biol. Chem. 253:5617-5624 (1978)).

In one embodiment, an acceptor for the sialyltransferase is present onthe glycopeptide to be modified upon in vivo synthesis of theglycopeptide. Such glycopeptides can be sialylated using the claimedmethods without prior modification of the glycosylation pattern of theglycopeptide. Alternatively, the methods of the invention can be used tosialylate a peptide that does not include a suitable acceptor; one firstmodifies the peptide to include an acceptor by methods known to those ofskill in the art. In an exemplary embodiment, a GalNAc residue is addedby the action of a GalNAc transferase.

In an exemplary embodiment, the acceptor is assembled by attaching agalactose residue to, for example, a GlcNAc or another appropriatesaccharide moiety that is linked to the peptide. The method includesincubating the peptide to be modified with a reaction mixture thatcontains a suitable amount of a galactosyltransferase (e.g., galβ1,3 orgalβ1,4), and a suitable galactosyl donor (e.g., UDP-galactose). Thereaction is allowed to proceed substantially to completion or,alternatively, the reaction is terminated when a preselected amount ofthe galactose residue is added. Other methods of assembling a selectedsaccharide acceptor will be apparent to those of skill in the art.

In yet another embodiment, glycopeptide-linked oligosaccharides arefirst “trimmed,” either in whole or in part, to expose either anacceptor for the sialyltransferase or a moiety to which one or moreappropriate residues can be added to obtain a suitable acceptor. Enzymessuch as glycosyltransferases and endoglycosidases (see, for example U.S.Pat. No. 5,716,812) are useful for the attaching and trimming reactions.

The examples set forth above provide an illustration of the power of themethods set forth herein. Using the methods of the invention, it ispossible to “trim back” and build up a carbohydrate residue ofsubstantially any desired structure. The modified sugar can be added tothe termini of the carbohydrate moiety as set forth above, or it can beintermediate between the peptide core and the terminus of thecarbohydrate.

In an exemplary embodiment, an existing sialic acid is removed from aglycopeptide using a sialidase, thereby unmasking all or most of theunderlying galactosyl residues. Alternatively, a peptide or glycopeptideis labeled with galactose residues, or an oligosaccharide residue thatterminates in a galactose unit. Following the exposure of or addition ofthe galactose residues, an appropriate sialyltransferase is used to adda modified sialic acid.

One can assess differences in glycosylation pattern not only bystructural analysis, but also by comparison of one or more biologicalactivities of the protein. The glycopeptide produced by the methods ofthe invention typically exhibit an improvement in one more biologicalactivities as compared to the unmodified glycopeptide. For example,glycopeptides of the invention can have greater therapeutic efficacy asmeasured by solubility, resistance to proteolytic attack and thermalinactivation, immunogenicity, half-life, bioactivity, stability and thelike. The amount of the improvement observed is preferably statisticallysignificant, and is more preferably at least about a 50% improvement,and still more preferably is at least about 80%.

i. Enzymes

1. Glycosyltransferases

Glycosyltransferases catalyze the addition of activated sugars (e.g.,donor NDP-sugars), in a step-wise fashion, to a protein, glycopeptide,lipid or glycolipid or to the non-reducing end of a growingoligosaccharide. N-linked glycopeptides are synthesized via atransferase and a lipid-linked oligosaccharide donor Dol-PP-NAG₂Glc₃Man₉in an en block transfer followed by trimming of the core. In this casethe nature of the “core” saccharide is somewhat different fromsubsequent attachments. A very large number of glycosyltransferases areknown in the art.

The glycosyltransferase to be used in the present invention may be anyas long as it can utilize a selected glycosyl donor moiety as a sugardonor. Examples of such enzymes include Leloir pathwayglycosyltransferase, such as gal actosyltransferase,N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase,fucosyltransferase, sialyltransferase, mannosyltransferase,xylosyltransferase, glucurononyltransferase and the like.

For enzymatic saccharide syntheses that involve glycosyltransferasereactions, glycosyltransferase can be cloned, or isolated from anysource. Many cloned glycosyltransferases are known, as are theirpolynucleotide sequences. See, e.g., Taniguchi et al., 2002, Handbook ofglycosyltransferases and related genes, Springer, Tokyo; and “The WWWGuide To Cloned Glycosyltransferases,” (available atwww.vei.co.uk/TGN/gt_guide.htm). Glycosyltransferase amino acidsequences and nucleotide sequences encoding glycosyltransferases fromwhich the amino acid sequences can be deduced are also found in variouspublicly available databases, including GenBank, Swiss-Prot, EMBL, andothers.

Glycosyltransferases that can be employed in the methods of theinvention include, but are not limited to, galactosyltransferases,fucosyltransferases, glucosyltransferases,N-acetylgalactosaminyltransferases, N-acetylglucosarninyltransferases,glucuronyltransferases, sialyltransferases, mannosyltransferases,glucuronic acid transferases, galactunoric acid transferases, andoligosaccharyltransferases. Suitable glycosyltransferases include thoseobtained from eukaryotes, as well as from prokaryotes.

A number of methods of using glycosyltransferases to synthesize desiredoligosaccharide structures are known and are generally applicable to theinstant invention. Exemplary methods are described, for instance, WO96/32491, Ito et al., Pure Appl. Chem. 65: 753 (1993), and U.S. Pat.Nos. 5,352,670, 5,374,541, and 5,545,553.

The present invention is practiced using a single glycosyltransferase ora combination of glycosyltransferases. For example, one can use acombination of a sialyltransferase and a galactosyltransferase. In thoseembodiments using more than one enzyme, the enzymes and substrates arepreferably combined in an initial reaction mixture, or the enzymes andreagents for a second enzymatic reaction are added to the reactionmedium once the first enzymatic reaction is complete or nearly complete.By conducting two enzymatic reactions in sequence in a single vessel,overall yields are improved over procedures in which an intermediatespecies is isolated. Moreover, cleanup and disposal of extra solventsand by-products is reduced.

DNA which encodes the enzyme glycosyltransferases may be obtained bychemical synthesis, by screening reverse transcripts of mRNA fromappropriate cells or cell line cultures, by screening genomic librariesfrom appropriate cells, or by combinations of these procedures.Screening of mRNA or genomic DNA may be carried out witholigonucleotides probes generated from the glycosyltransferases genesequence. Probes may be labeled with a detectable group such as afluorescent group, a radioactive atom or a chemiluminescent group inaccordance with known procedures and used in conventional hybridizationassays. In the alternative, glycosyltransferases gene sequences may beobtained by use of the polymerase chain reaction (PCR) procedure, withthe PCR oligonucleotides primers being produced from theglycosyltransferases gene sequence. See, U.S. Pat. No. 4,683,195 toMullis et al. and U.S. Pat. No. 4,683,202 to Mullis.

The glycosyltransferases enzyme may be synthesized in host cellstransformed with vectors containing DNA encoding theglycosyltransferases enzyme. A vector is a replicable DNA construct.Vectors are used either to amplify DNA encoding the glycosyltransferasesenzyme and/or to express DNA which encodes the glycosyltransferasesenzyme. An expression vector is a replicable DNA construct in which aDNA sequence encoding the glycosyltransferases enzyme is operably linkedto suitable control sequences capable of effecting the expression of theglycosyltransferases enzyme in a suitable host. The need for suchcontrol sequences will vary depending upon the host selected and thetransformation method chosen. Generally, control sequences include atranscriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences which control the termination of transcription andtranslation. Amplification vectors do not require expression controldomains. All that is needed is the ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transform ants.

a) Fucosyltransferases

In some embodiments, a glycosyltransferase used in the method of theinvention is a fucosyltransferase. Fucosyltransferases are known tothose of skill in the art. Exemplary fucosyltransferases includeenzymes, which transfer L-fucose from GDP-fucose to a hydroxy positionof an acceptor sugar. Fucosyltransferases that transfer non-nucleotidesugars to an acceptor are also of use in the present invention.

In some embodiments, the acceptor sugar is, for example, the GlcNAc in aGalβ(1→3,4)GlcNAcβ-group in an oligosaccharide glycoside. Suitablefucosyltransferases for this reaction include theGalβ(1→3,4)GlcNAcβ1-α(1→3,4)fucosyltransferase (FTIII E.C. No.2.4.1.65), which was first characterized from human milk (see, Palcic,et al., Carbohydrate Res. 190: 1-11 (1989); Prieels, et al., J. Biol.Chem. 256: 10456-10463 (1981); and Nunez, et al., Can. J. Chem. 59:2086-2095 (1981)) and the Galβ(1→4)GlcNAcβ-α-fucosyltransferases (FTIV,FTV, FTVI) which are found in human serum. FTVII (E.C. No. 2.4.1.65), asialyl α(2→3)Galβ(1→3) GlcNAcβ fucosyltransferase, has also beencharacterized. A recombinant form of the Galβ(1→3,4)GlcNAcβ-α(1→3,4)fucosyltransferase has also been characterized (see,Dumas, et al., Bioorg. Med. Letters 1: 425-428 (1991) andKukowska-Latallo, et al., Genes and Development 4: 1288-1303 (1990)).Other exemplary fucosyltransferases include, for example, α1,2fucosyltransferase (E.C. No. 2.4.1.69). Enzymatic fucosylation can becarried out by the methods described in Mollicone, et al., Eur. J.Biochem. 191: 169-176 (1990) or U.S. Pat. No. 5,374,655. Cells that areused to produce a fucosyltransferase will also include an enzymaticsystem for synthesizing GDP-fucose.

b) Galactosyltransferases

In another group of embodiments, the glycosyltransferase is agalactosyltransferase. Exemplary galactosyltransferases include α(1,3)galactosyltransferases (E.C. No. 2.4.1.151, see, e.g., Dabkowski et al.,Transplant Proc. 25:2921 (1993) and Yamamoto et al. Nature 345: 229-233(1990), bovine (GenBank j04989, Joziasse et al., J. Biol. Chem. 264:14290-14297 (1989)), murine (GenBank m26925; Larsen et al., Proc. Nat'l.Acad. Sci. USA 86: 8227-8231 (1989)), porcine (GenBank L36152; Strahanet al., Immunogenetics 41: 101-105 (1995)). Another suitable α1,3galactosyltransferase is that which is involved in synthesis of theblood group B antigen (EC 2.4.1.37, Yamamoto et al., J. Biol. Chem. 265:1146-1151 (1990) (human)).

Also suitable for use in the methods of the invention are β(1,4)galactosyltransferases, which include, for example, EC 2.4.1.90 (LacNAcsynthetase) and EC 2.4.1.22 (lactose synthetase) (bovine (D'Agostaro etal., Eur. J. Biochem. 183: 211-217 (1989)), human (Masri et al.,Biochem. Biophys. Res. Commun. 157: 657-663 (1988)), murine (Nakazawa etal., J. Biochem. 104: 165-168 (1988)), as well as E.C. 2.4.1.38 and theceramide galactosyltransferase (EC 2.4.1.45, Stahl et al., J. Neurosci.Res. 38: 234-242 (1994)). Other suitable galactosyltransferases include,for example, α1,2 galactosyltransferases (from e.g., Schizosaccharomycespombe, Chapell et al., Mol Biol. Cell 5: 519-528 (1994)).

The production of proteins such as the enzyme GalNAc T_(1-XX) fromcloned genes by genetic engineering is well known. See, e.g., U.S. Pat.No. 4,761,371. One method involves collection of sufficient samples,then the amino acid sequence of the enzyme is determined by N-terminalsequencing. This information is then used to isolate a cDNA cloneencoding a full-length (membrane bound) transferase which uponexpression in the insect cell line Sf9 resulted in the synthesis of afully active enzyme. The acceptor specificity of the enzyme is thendetermined using a semiquantitative analysis of the amino acidssurrounding known glycosylation sites in 16 different proteins followedby in vitro glycosylation studies of synthetic peptides. This work hasdemonstrated that certain amino acid residues are overrepresented inglycosylated peptide segments and that residues in specific positionssurrounding glycosylated serine and threonine residues may have a moremarked influence on acceptor efficiency than other amino acid moieties.

c) Sialyltransferases

Sialyltransferases are another type of glycosyltransferase that isuseful in the recombinant cells and reaction mixtures of the invention.Cells that produce recombinant sialyltransferases will also produceCMP-sialic acid, which is a sialic acid donor for sialyltransferases.Examples of sialyltransferases that are suitable for use in the presentinvention include ST3Gal III (e.g., a rat or human ST3Gal III), ST3GalIV, ST3Gal I, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, ST6GalNAc II,and ST6GalNAc III (the sialyltransferase nomenclature used herein is asdescribed in Tsuji et al., Glycobiology 6: v-xiv (1996)). An exemplaryα(2,3)sialyltransferase referred to as α(2,3)sialyltransferase (EC2.4.99.6) transfers sialic acid to the non-reducing terminal Gal of aGalβ1→3Glc disaccharide or glycoside. See, Van den Eijnden et al., J.Biol. Chem. 256: 3159 (1981), Weinstein et al., J. Biol. Chem. 257:13845 (1982) and Wen et al., J. Biol. Chem. 267: 21011 (1992). Anotherexemplary α2,3-sialyltransferase (EC 2.4.99.4) transfers sialic acid tothe non-reducing terminal Gal of the disaccharide or glycoside. see,Rearick et al., J. Biol. Chem. 254: 4444 (1979) and Gillespie et al., J.Biol. Chem. 267: 21004 (1992). Further exemplary enzymes includeGal-β-1,4-GlcNAc α-2,6 sialyltransferase (See, Kurosawa et al. Eur. J.Biochem. 219: 375-381 (1994)).

Preferably, for glycosylation of carbohydrates of glycopeptides thesialyltransferase will be able to transfer sialic acid to the sequenceGalβ1,4GlcNAc-, the most common penultimate sequence underlying theterminal sialic acid on fully sialylated carbohydrate structures (see,Table 2).

TABLE 2 Sialyltransferases which use the Galβ1,4GlcNAc sequence as anacceptor substrate. Sialyltransferase Source Sequence(s) formed Ref.ST6Gal I Mammalian NeuAcI2,6Galβ1,4GlCNAc- 1 ST3Gal III MammalianNeuAcI2,3Galβ1,4GlCNAc- 1 NeuAcI2,3Galβ1,3GlGNAc- ST3Gal IV MammalianNeuAcI2,3Galβ1,4GlCNAc- 1 NeuAcI2,3Galβ1,3GlCNAc- ST6Gal II MammalianNeuAcI2,6Galβ1,4GlCNA ** ST6Gal II photobacteriumNeuAcI2,6Galβ1,4GlCNAc- 2 ST3Gal V N. meningitidesNeuAcI2,3Galβ1,4GlCNAc- 3 N. gonorrhoeae 1) Goochee et al.,Bio/Technology 9: 1347-1355 (1991) 2) Yamamoto et al., J. Biochem. 120:104-110 (1996) 3) Gilbert et al., J. Biol. Chem. 271: 28271-28276 (1996)

An example of a sialyltransferase that is useful in the claimed methodsis ST3Gal III, which is also referred to as α(2,3)sialyltransferase (EC2.4.99.6). This enzyme catalyzes the transfer of sialic acid to the Galof a Galβ1,3GlcNAc or Galβ1,4GlcNAc glycoside (see, e.g., Wen et al., J.Biol. Chem. 267: 21011 (1992); Van den Eijnden et al., J. Biol. Chem.256: 3159 (1991)) and is responsible for sialylation ofasparagine-linked oligosaccharides in glycopeptides. The sialic acid islinked to a Gal with the formation of an α-linkage between the twosaccharides. Bonding (linkage) between the saccharides is between the2-position of NeuAc and the 3-position of Gal. This particular enzymecan be isolated from rat liver (Weinstein et al., J. Biol. Chem. 257:13845 (1982)); the human cDNA (Sasaki et al. (1993) J. Biol. Chem. 268:22782-22787; Kitagawa & Paulson (1994) J. Biol. Chem. 269: 1394-1401)and genomic (Kitagawa et al. (1996) J. Biol. Chem. 271: 931-938) DNAsequences are known, facilitating production of this enzyme byrecombinant expression. In a preferred embodiment, the claimedsialylation methods use a rat ST3Gal III.

Other exemplary sialyltransferases of use in the present inventioninclude those isolated from Campylobacter jejuni, including the α(2,3).See, e.g, WO99/49051.

Other sialyltransferases, including those listed in Table 4, are alsouseful in an economic and efficient large-scale process for sialylationof commercially important glycopeptides. As a simple test to find outthe utility of these other enzymes, various amounts of each enzyme(1-100 mU/mg protein) are reacted with asialo-α₁ AGP (at 1-10 mg/ml) tocompare the ability of the sialyltransferase of interest to sialylateglycopeptides relative to either bovine ST6Gal I, ST3Gal III or bothsialyltransferases. Alternatively, other glycopeptides or glycopeptides,or N-linked oligosaccharides enzymatically released from the peptidebackbone can be used in place of asialo-α₁ AGP for this evaluation.Sialyltransferases with the ability to sialylate N-linkedoligosaccharides of glycopeptides more efficiently than ST6Gal I areuseful in a practical large-scale process for peptide sialylation (asillustrated for ST3Gal III in this disclosure).

d) Other Glycosyltransferases

One of skill in the art will understand that other glycosyltransferasescan be substituted into similar transferase cycles as have beendescribed in detail for the sialyltransferase. In particular, theglycosyltransferase can also be, for instance, glucosyltransferases,e.g., Alg8 (Stagljov et al., Proc. Natl. Acad. Sci. USA 91: 5977 (1994))or Alg5 (Heesen et al., Eur. J. Biochem. 224: 71 (1994)).

N-acetylgalactosaminyltransferases are also of use in practicing thepresent invention. Suitable N-acetylgalactosaminyltransferases include,but are not limited to, α(1,3) N-acetylgalactosaminyltransferase, β(1,4)N-acetylgalactosaminyltransferases (Nagata et al., J. Biol. Chem. 267:12082-12089 (1992) and Smith et al., J. Biol. Chem. 269: 15162 (1994))and polypeptide N-acetylgalactosaminyltransferase (Homa et al., J. Biol.Chem. 268: 12609 (1993)). Suitable N-acetylglucosaminyltransferasesinclude GnTI (2.4.1.101, Hull et al., BBRC 176: 608 (1991)), GnTII,GnTIII (Ihara et al., J. Biochem. 113: 692 (1993)), GnTIV, and GnTV(Shoreiban et al., J. Biol. Chem. 268: 15381 (1993)), O-linkedN-acetylglucosaminyltransferase (Bierhuizen et al., Proc. Natl. Acad.Sci. USA 89: 9326 (1992)), N-acetylglucosamine-1-phosphate transferase(Rajput et al., Biochem J. 285: 985 (1992), and hyaluronan synthase.

Mannosyltransferases are of use to transfer modified mannose moieties.Suitable mannosyltransferases include α(1,2) mannosyltransferase, α(1,3)mannosyltransferase, α(1,6) mannosyltransferase, α(1,4)mannosyltransferase, Dol-P-Man synthase, OCh1, and Pmt1 (see, Kornfeldet al., Annu. Rev. Biochem. 54: 631-664 (1985)).

Xylosyltransferases are also useful in the present invention. See, forexample, Rodgers, et al., Biochem. J., 288:817-822 (1992); and Elbain,et al., U.S. Pat. No., 6,168,937.

Other suitable glycosyltransferase cycles are described in Ichikawa etal., JACS 114: 9283 (1992), Wong et al., J. Org. Chem. 57: 4343 (1992),and Ichikawa et al. in CARBOHYDRATES AND CARBOHYDRATE POLYMERS. Yaltami,ed. (ATL Press, 1993).

Prokaryotic glycosyltransferases are also useful in practicing theinvention. Such glycosyltransferases include enzymes involved insynthesis of lipooligosaccharides (LOS), which are produced by many gramnegative bacteria. The LOS typically have terminal glycan sequences thatmimic glycoconjugates found on the surface of human epithelial cells orin host secretions (Preston et al., Critical Reviews in Microbiology23(3): 139-180 (1996)). Such enzymes include, but are not limited to,the proteins of the rfa operons of species such as E. coli andSalmonella typhimurium, which include a β1,6 galactosyltransferase and aβ1,3 galactosyltransferase (see, e.g., EMBL Accession Nos. M80599 andM86935 (E. coli); EMBL Accession No. S56361 (S. typhimurium)), aglucosyltransferase (Swiss-Prot Accession No. P25740 (E. coli), anβ1,2-glucosyltransferase (rfaJ) (Swiss-Prot Accession No. P27129 (E.coli) and Swiss-Prot Accession No. P19817 (S. typhimurium)), and anβ1,2-N-acetylglucosaminyltransferase (rfaK) (EMBL Accession No. U00039(E. coli). Other glycosyltransferases for which amino acid sequences areknown include those that are encoded by operons such as rfaB, which havebeen characterized in organisms such as Klebsiella pneumoniae, E. coli,Salmonella typhimurium, Salmonella enterica, Yersinia enterocolitica,Mycobacterium leprosum, and the rh1 operon of Pseudomonas aeruginosa.

Also suitable for use in the present invention are glycosyltransferasesthat are involved in producing structures containinglacto-N-neotetraose,D-galactosyl-β-1,4-N-acetyl-D-glucosaminyl-β-1,3-D-galactosyl-β-1,4-D-glucose,and the P_(k) blood group trisaccharide sequence,D-galactosyl-α-1,4-D-galactosyl-β-1,4-D-glucose, which have beenidentified in the LOS of the mucosal pathogens Neisseria gonnorhoeae andN. meningitidis (Scholten et al., J. Med. Microbiol. 41: 236-243(1994)). The genes from N. meningitidis and N. gonorrhoeae that encodethe glycosyltransferases involved in the biosynthesis of thesestructures have been identified from N. meningitidis immunotypes L3 andL1 (Jennings et al., Mol. Microbiol. 18: 729-740 (1995)) and the N.gonorrhoeae mutant F62 (Gotshlich, J. Exp. Med. 180: 2181-2190 (1994)).In N. meningitidis, a locus consisting of three genes, IgtA, IgtB andIgE, encodes the glycosyltransferase enzymes required for addition ofthe last three of the sugars in the lacto-N-neotetraose chain (Wakarchuket al., J. Biol. Chem. 271: 19166-73 (1996)). Recently the enzymaticactivity of the IgtB and IgtA gene product was demonstrated, providingthe first direct evidence for their proposed glycosyltransferasefunction (Wakarchuk et al., J. Biol. Chem. 271(45): 28271-276 (1996)).In N. gonorrhoeae, there are two additional genes, IgtD which adds.beta.-D-GalNAc to the 3 position of the terminal galactose of thelacto-N-neotetraose structure and IgtC which adds a terminal α-D-Gal tothe lactose element of a truncated LOS, thus creating the P_(k) bloodgroup antigen structure (Gotshlich (1994), supra.). In N. meningitidis,a separate immunotype L1 also expresses the P_(k) blood group antigenand has been shown to carry an IgtC gene (Jennings et al., (1995),supra.). Neisseria glycosyltransferases and associated genes are alsodescribed in U.S. Pat. No. 5,545,553 (Gotschlich). Genes forα1,2-fucosyltransferase and α1,3-fucosyltransferase from Helicobacterpylori has also been characterized (Martin et al., J. Biol. Chem. 272:21349-21356 (1997)). Also of use in the present invention are theglycosyltransferases of Campylobacter jejuni (see, for example,afmb.cnrs-mrs.fr/˜pedro/CAZY/gtf_(—)42.html).

2. Sulfotransferases

The invention also provides methods for producing peptides that includesulfated molecules, including, for example sulfated polysaccharides suchas heparin, heparan sulfate, carragenen, and related compounds. Suitablesulfotransferases include, for example, chondroitin-6-sulphotransferase(chicken cDNA described by Fukuta et al., J. Biol. Chem. 270:18575-18580 (1995); GenBank Accession No. D49915), glycosaminoglycanN-acetylglucosamine N-deacetylase/N-sulfotransferase 1 (Dixon et al.,Genomics 26: 239-241 (1995); UL18918), and glycosaminoglycanN-acetylglucosamine N-deacetylase/N-sulfotransferase 2 (murine cDNAdescribed in Orellana et al., J. Biol. Chem. 269: 2270-2276 (1994) andEriksson et al., J. Biol. Chem. 269: 10438-10443 (1994); human cDNAdescribed in GenBank Accession No. U2304).

3. Cell-Bound Glycosyltransferases

In another embodiment, the enzymes utilized in the method of theinvention are cell-bound glycosyltransferases. Although many solubleglycosyltransferases are known (see, for example, U.S. Pat. No.5,032,519), glycosyltransferases are generally in membrane-bound formwhen associated with cells. Many of the membrane-bound enzymes studiedthus far are considered to be intrinsic proteins; that is, they are notreleased from the membranes by sonication and require detergents forvolatilization. Surface glycosyltransferases have been identified on thesurfaces of vertebrate and invertebrate cells, and it has also beenrecognized that these surface transferases maintain catalytic activityunder physiological conditions. However, the more recognized function ofcell surface glycosyltransferases is for intercellular recognition(Roth, MOLECULAR APPROACHES to SUPRACELLULAR PHENOMENA, 1990).

Methods have been developed to alter the glycosyltransferases expressedby cells. For example, Larsen et al., Proc. Natl. Acad. Sci. USA 86:8227-8231 (1989), report a genetic approach to isolate cloned cDNAsequences that determine expression of cell surface oligosaccharidestructures and their cognate glycosyltransferases. A cDNA librarygenerated from mRNA isolated from a murine cell line known to expressUDP-galactose:.β-D-galactosyl-1,4-N-acetyl-D-glucosaminideα-1,3-galactosyltransferase was transfected into COS-1 cells. Thetransfected cells were then cultured and assayed for α1-3galactosyltransferase activity.

Francisco et al., Proc. Natl. Acad. Sci. USA 89: 2713-2717 (1992),disclose a method of anchoring β-lactamase to the external surface ofEscherichia coli. A tripartite fusion consisting of (i) a signalsequence of an outer membrane protein, (ii) a membrane-spanning sectionof an outer membrane protein, and (iii) a complete mature β-lactamasesequence is produced resulting in an active surface bound β-lactamasemolecule. However, the Francisco method is limited only to procaryoticcell systems and as recognized by the authors, requires the completetripartite fusion for proper functioning.

4. Fusion Proteins

In other exemplary embodiments, the methods of the invention utilizefusion proteins that have more than one enzymatic activity that isinvolved in synthesis of a desired glycopeptide conjugate. The fusionpolypeptides can be composed of, for example, a catalytically activedomain of a glycosyltransferase that is joined to a catalytically activedomain of an accessory enzyme. The accessory enzyme catalytic domaincan, for example, catalyze a step in the formation of a nucleotide sugarwhich is a donor for the glycosyltransferase, or catalyze a reactioninvolved in a glycosyltransferase cycle. For example, a polynucleotidethat encodes a glycosyltransferase can be joined, in-frame, to apolynucleotide that encodes an enzyme involved in nucleotide sugarsynthesis. The resulting fusion protein can then catalyze not only thesynthesis of the nucleotide sugar, but also the transfer of the sugarmoiety to the acceptor molecule. The fusion protein can be two or morecycle enzymes linked into one expressible nucleotide sequence. In otherembodiments the fusion protein includes the catalytically active domainsof two or more glycosyltransferases. See, for example, U.S. Pat. No.5,641,668. The modified glycopeptides of the present invention can bereadily designed and manufactured utilizing various suitable fusionproteins (see, for example, PCT Patent Application PCT/CA98/01180, whichwas published as WO 99/31224 on Jun. 24, 1999.)

Protein Remodeling and Purification

The methods presented herein can be practiced in any useful order onpeptides and glycopeptides that are in crude form, e.g., as expressed,are partially purified or are fully purified. For example, in oneembodiment, a peptide or glycopeptide is expressed, purified, remodeledusing a method of the invention and subsequently purified. In anotherexemplary embodiment, a peptide or glycopeptide is expressed, andisolated in crude form. The crude material is remodeled using a methodof the invention and the remodeled peptide or glycopeptide is purified.In yet another exemplary embodiment, the expressed peptide orglycopeptide is partially purified, e.g, to remove cellular debris,remodeled and subsequently purified. Other variations on these schemeswill be apparent to those of skill in the art and they are within thescope of the present invention.

Purification of Peptide Conjugates and Oligosaccharides

a. Oligosaccharide Purification

The reagent oligosaccharides produced by the above processes can be usedwithout purification. However, it is usually preferred to recover theproduct. Standard, well known techniques for recovery of glycosylatedsaccharides such as thin or thick layer chromatography, columnchromatography, ion exchange chromatography, or membrane filtration canbe used. It is preferred to use membrane filtration, more preferablyutilizing a reverse osmotic membrane, or one or more columnchromatographic techniques for the recovery as is discussed hereinafterand in the literature cited herein. For instance, membrane filtrationwherein the membranes have molecular weight cutoff of about 3000 toabout 10,000 can be used to remove proteins such as glycosyltransferases. Nanofiltration or reverse osmosis can then be used toremove salts and/or purify the product saccharides (see, e.g., WO98/15581). Nanofilter membranes are a class of reverse osmosis membraneswhich pass monovalent salts but retain polyvalent salts and unchargedsolutes larger than about 100 to about 4,000 Daltons, depending upon themembrane used. Thus, in a typical application, saccharides prepared bythe methods of the present invention will be retained in the membraneand contaminating salts will pass through. Additional purificationtechniques include recrystallization, chromatography (silica, reversedphase, ion exchange) and precipitation.

b. Protein (Glycoprotein) Purification

If the modified glycopeptide is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration; optionally,the protein may be concentrated with a commercially available proteinconcentration filter, followed by separating the polypeptide variantfrom other impurities by one or more steps selected from immunoaffinitychromatography, ion-exchange column fractionation (e.g., ondiethylaminoethyl (DEAE) or matrices containing carboxymethyl orsulfopropyl groups), chromatography on Blue-Sepharose, CMBlue-Sepharose, MONO-Q, MONO-S, lentil lectin-Sepharose, WGA-Sepharose,Con A-Sepharose, Ether Toyopearl, Butyl Toyopearl, Phenyl Toyopearl, orprotein A Sepharose, SDS-PAGE chromatography, silica chromatography,chromatofocusing, reverse phase HPLC (e.g., silica gel with appendedaliphatic groups), gel filtration using, e.g., Sephadex molecular sieveor size-exclusion chromatography, chromatography on columns thatselectively bind the polypeptide, and ethanol or ammonium sulfateprecipitation.

Modified glycopeptides produced in culture are usually isolated byinitial extraction from cells, enzymes, etc., followed by one or moreconcentration, salting-out, aqueous ion-exchange, or size-exclusionchromatography steps. Additionally, the modified glycopeptide may bepurified by affinity chromatography. Finally, HPLC may be employed forfinal purification steps.

A protease inhibitor, e.g., methylsulfonylfluoride (PMSF) may beincluded in any of the foregoing steps to inhibit proteolysis andantibiotics may be included to prevent the growth of adventitiouscontaminants.

Within another embodiment, supernatants from systems which produce themodified glycopeptide of the invention are first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate may be applied to a suitablepurification matrix. For example, a suitable affinity matrix maycomprise a ligand for the peptide, a lectin or antibody molecule boundto a suitable support. Alternatively, an anion-exchange resin may beemployed, for example, a matrix or substrate having pendant DEAE groups.Suitable matrices include acrylamide, agarose, dextran, cellulose, orother types commonly employed in protein purification. Alternatively, acation-exchange step may be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are particularly preferred.

Finally, one or more RP-HPLC steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, may beemployed to further purify a polypeptide variant composition. Some orall of the foregoing purification steps, in various combinations, canalso be employed to provide a homogeneous modified glycopeptide.

The modified glycopeptide of the invention resulting from a large-scalefermentation may be purified by methods analogous to those disclosed byUrdal et al., J. Chromatog. 296: 171 (1984). This reference describestwo sequential, RP-HPLC steps for purification of recombinant human IL-2on a preparative HPLC column. Alternatively, techniques such as affinitychromatography, may be utilized to purify the modified glycopeptide.These include methods using antibodies, cofactors, substrates or othersmall molecule agent that selectively binds to the protein of interest.

Affinity tags on the mutant amidase to allow for the simple removal fromthe reaction mixture.

The Compositions

In another aspect, the present invention provides compositions ofglycopeptides prepared by the method of the invention. Using the methodsof the invention, it is possible to substantially completely remodel aparticular glycosyl residue on a glycopeptide. Thus, in an exemplaryembodiment, the invention provides a glycopeptide in which at leastabout 80% of a population of a selected acceptor moiety on theglycopeptide is glycosylated with the glycosyl residue added by themutant amidase.

Numerous reaction formats, e.g., solid phase and solution methodologies,will suggest themselves. In an exemplary embodiment, the method of theinvention is used to produce a glycopeptide that is attached to a solidsupport.

The amino acid sequence of the glycopeptides of the invention can beeither full-length or truncated. Exemplary proteins include interferonbeta, interferon omega, enbrel, EPO, NESP, FSH and the Blood Factors(VIIa, IX, VIII).

Pharmaceutical Formulations

The compounds produced by the methods of the invention can then be usedin a variety of applications, e.g., as antigens, diagnostic reagents, oras therapeutics. Thus, the present invention also providespharmaceutical compositions which can be used in treating a variety ofconditions. The pharmaceutical compositions are comprised ofglycopeptides made according to the methods described above.

Pharmaceutical compositions of the invention are suitable for use in avariety of drug delivery systems. Suitable formulations for use in thepresent invention are found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985). For a briefreview of methods for drug delivery, see, Langer, Science 249:1527-1533(1990).

The pharmaceutical compositions may be formulated for any appropriatemanner of administration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268 and 5,075,109.

Commonly, the pharmaceutical compositions are administered parenterally,e.g., intravenously or subcutaneous. Thus, the invention providescompositions for parenteral administration which comprise the compounddissolved or suspended in an acceptable carrier, preferably an aqueouscarrier, e.g., water, buffered water, saline, PBS and the like. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents, detergents and the like. Exemplary buffers include phosphate,histidine, glycine and combinations thereof which can also containexcepients such as sugars (i.e. trehalose, mannose, sucrose, glucose,galactose and sialic acid), salts (i.e. sodium chloride, potassiumchloride, magnesium salts, calcium salts), proteins (i.e. albumin),detergents (i.e. polysorbate 80) and preservatives (i.e. sodiumbenzoate).

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably from 5 to 9 and most preferably from 7 and 8. Insome embodiments the glycopeptides of the invention can be incorporatedinto liposomes formed from standard vesicle-forming lipids. A variety ofmethods are available for preparing liposomes, as described in, e.g.,Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos.4,235,871, 4,501,728 and 4,837,028. The targeting of liposomes using avariety of targeting agents (e.g., the sialyl galactosides of theinvention) is well known in the art (see, e.g., U.S. Pat. Nos. 4,957,773and 4,603,044).

Standard methods for coupling targeting agents to liposomes can be used.These methods generally involve incorporation into liposomes of lipidcomponents, such as phosphatidylethanolamine, which can be activated forattachment of targeting agents, or derivatized lipophilic compounds,such as lipid derivatized glycopeptides of the invention.

Targeting mechanisms generally require that the targeting agents bepositioned on the surface of the liposome in such a manner that thetarget moieties are available for interaction with the target, forexample, a cell surface receptor. The carbohydrates of the invention maybe attached to a lipid molecule before the liposome is formed usingmethods known to those of skill in the art (e.g., alkylation oracylation of a hydroxyl group present on the carbohydrate with a longchain alkyl halide or with a fatty acid, respectively). Alternatively,the liposome may be fashioned in such a way that a connector portion isfirst incorporated into the membrane at the time of forming themembrane. The connector portion must have a lipophilic portion which isfirmly embedded and anchored in the membrane. It must also have areactive portion which is chemically available on the aqueous surface ofthe liposome. The reactive portion is selected so that it will bechemically suitable to form a stable chemical bond with the targetingagent or carbohydrate which is added later. In some cases it is possibleto attach the target agent to the connector molecule directly, but inmost instances it is more suitable to use a third molecule to act as achemical bridge, thus linking the connector molecule which is in themembrane with the target agent or carbohydrate which is extended, threedimensionally, off of the vesicle surface.

The blood-residency of therapeutic glycopeptides can also be enhancedwith polyethylene glycol (PEG). Chemical modification of proteins withPEG (PEGylation) increases their molecular size and steric hindrance,both of which are dependent on the PEG attached to the protein. Thisresults in an improvement of plasma half-lives and inproteolytic-stability, and a decrease in immunogenicity and hepaticuptake (Chaffee et al. J. Clin. Invest. 89:1643-1651 (1992); Pyatak etal. Res. Commun. Chem. Pathol Pharmacol. 29:113-127 (1980)). PEGylationof interleukin-2 has been reported to increase its antitumor potency invivo (Katre et al. Proc. Natl. Acad. Sci. USA. 84:1487-1491 (1987)) andPEGylation of an F (ab′)2 derived from the monoclonal antibody A7 hasimproved its tumor localization (Kitamura et al. Biochem. Biophys. Res.Commun. 28:1387-1394 (1990)).

The compositions containing the glycopeptides can be administered forprophylactic and/or therapeutic treatments. In therapeutic applications,compositions are administered to a patient already suffering from adisease, as described above, in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications. Anamount adequate to accomplish this is defined as a “therapeuticallyeffective dose.” Amounts effective for this use will depend on theseverity of the disease and the weight and general state of the patient,but generally range from about 0.5 mg to about 2,000 mg of glycopeptideper day for a 70 kg patient, with dosages of from about 5 mg to about200 mg of the compounds per day being more commonly used.

In prophylactic applications, compositions containing the glycopeptidesof the invention are administered to a patient susceptible to orotherwise at risk of a particular disease. Such an amount is defined tobe a “prophylactically effective dose.” In this use, the precise amountsagain depend on the patient's state of health and weight, but generallyrange from about 0.5 mg to about 1,000 mg per 70 kilogram patient, morecommonly from about 5 mg to about 200 mg per 70 kg of body weight.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of the glycopeptides of this invention sufficient toeffectively treat the patient.

The glycopeptides may also find use as diagnostic reagents. For example,labeled compounds can be used to locate areas of inflammation or tumormetastasis in a patient suspected of having an inflammation. For thisuse, the compounds can be labeled with appropriate radioisotopes, forexample, ¹²⁵I, ¹⁴C, or tritium.

The glycopeptides of the invention can be used as an immunogen for theproduction of monoclonal or polyclonal antibodies specifically reactivewith the compounds of the invention. The multitude of techniquesavailable to those skilled in the art for production and manipulation ofvarious immunoglobulin molecules can be used in the present invention.Antibodies may be produced by a variety of means well known to those ofskill in the art.

The production of non-human monoclonal antibodies, e.g., murine,lagomorpha, equine, etc., is well known and may be accomplished by, forexample, immunizing the animal with a preparation containing theglycopeptide of the invention. Antibody-producing cells obtained fromthe immunized animals are immortalized and screened, or screened firstfor the production of the desired antibody and then immortalized. For adiscussion of general procedures of monoclonal antibody production seeHarlow and Lane, Antibodies, A Laboratory Manual Cold Spring HarborPublications, N.Y. (1988).

The following examples are offered to illustrate, but not to limit thepresent invention.

EXAMPLE 1

This example describes the modification of PNGase-F for use in themethods of the invention.

PNGase F is a 34.7 kDa amidohydrolase secreted by Flavobacteriummeningosepticum. The enzyme hydrolyses N-linked oligosaccharide chainsof glycopeptides, converting the asparagine to aspartic acid with therelease of ammonia and the intact oligosaccharide chain. Enzymaticactivity of PNGase F requires recognition of both the peptide andcarbohydrate components of the substrate.

The catalytic mechanism for the mutant enzyme can catalyze either of twopathways (FIG. 2). Pathway A facilitates the synthesis of theoligosaccharide-protein bound using the reverse reaction of a peptidasehydrolysis step. The oligosaccharide contains an amino glycoside or aspecific or complex mixture of oligosaccharide structures recognized bythe enzyme. Pathway B utilizes an activated oligosaccharide and proceedswith addition of the sugar to Asn residues of the protein. The activatedsugar can contain F, Asn, Asn-peptide or other leaving group at thereducing termini. The activated sugar could also contain the1,2-oxazoline of GlcNAc at the reducing sugar.

Mutation of the PNGase-F protein is carried out as described above. Theknown active site residues include Asp (60), Glu (206), Glu (118), Trp(120), Arg (248) and His (193). One or more of these amino acids ismodified to improve the synthetic ability of the enzyme.

In another example, each of the three acidic residues, Asp (60), Glu(206) and Glu (118), are modified, (see Kuhn et al, J. Biol. Chem.270:9493 (1995)). All three residues are in the active site of theenzyme and in contact directly or indirectly with the bound sugar. Anexample of a modification is to change each residue to Asn or Ser.

By using modified PNGase-F, N-linked oligosaccharide structures can beadded to any protein of interest whether the protein already hasoligosaccharide structures or not. The only requirement is for theprotein of interest to contain a peptide sequence recognized by theimproved PNGase-F to allow oligosaccharide transfer. Because one cancontrol which oligosaccharide structure is used during proteinremodeling as well as the purity of the protein to be remodeled, a newglycopeptide product can be produced with a well defined andquantifiable structure.

EXAMPLE 2 2.1 Introduction of a Bi-Antennary-N-Linked Glycan ontoInterferon Beta

A solution of the mutated PNGase F (40,000 Units) is added to a solutionof E. coli produced interferon beta (0.35 mmol) dissolved in 100 mL ofphosphate buffer (250 mM) at pH 7.5, 0.2% polysorbate 80 andbiantennary-glycan (see FIG. 2). The solution is mixed at roomtemperature. To monitor the reaction, a small aliquot of the reaction isdiluted with the appropriate buffer and an IEF gel performed. When thereaction is complete, the reaction mixture is applied to a HIC column(C-4) and a gradient elution performed using a mixture of water andacetonitrile with a low percentage of TFA. Appropriate fractions arecombined, polysorbate 80 is added and the pH is adjusted to 7.4. Thebuffer is exchanged and the solution is concentrated by diafiltrationusing a 10 K membrane and exchanging against PBS buffer containingpolysorbate 80. The product of the reaction is analyzed using SDS PAGEand IEF analysis according to the procedures and reagents supplied byInvitrogen. Samples of native and glycopeptide are dialyzed againstwater and analyzed by MALDI-TOF MS.

2.2 Introduction of Tetra-Antennary-N-Linked-Glycans onto Erythropoietin

A solution of the mutated N-glycosidase A (1,000,000 Units) is added toa solution of E. coli produced erythropoietin (0.35 mmol) dissolved in 1L of phosphate/citrate buffer (250 mM) at pH 6.5, 0.02% polysorbate 80and tetra-antennary-glycan (see FIG. 2). The solution is mixed at roomtemperature. To monitor the reaction, a small aliquot of the reaction isdiluted with the appropriate buffer and a IEF gel performed. When thereaction is complete, the reaction mixture is applied to a HIC column(C-4) and a gradient elution performed using a mixture of water andacetonitrile with a low percentage of TFA. Appropriate fractions arecombined, polysorbate 80 is added and the pH adjusted to 7.4. The bufferis exchanged and the solution concentrated by diafiltration using a 10 Kmembrane and exchanging against PBS buffer containing polysorbate 80.The product of the reaction is analyzed using SDS PAGE and IEF analysisaccording to the procedures and reagents supplied by Invitrogen. Samplesof native and glycopeptide are dialyzed against water and analyzed byMALDI-TOF MS.

2.3 Preparation of Bi-Antennary-Glycan-F (FIG. 2B.1)

The biantennary-N-linked glycan isolated from egg protein (0.5 g) isadded to a solution containing pyridine (20 mL) and DMAP (0.1 g). Thesolution is cooled to 0° C., and acetic anhydride (400 mole eq) isslowly added. The reaction is warmed to 40° C. until the reaction iscompleted as determined by TLC. The reaction mixture is concentrated todryness and ethyl acetate is added to dissolve the residue. The organiclayer is washed with water, sat. sodium bicarbonate/water, water and wasthen dried (Na₂SO₄). After filtration, the filtrate is concentrated todryness and chromatography (silica) performed on the residue.Appropriate fractions were collected, concentrated and characterized byNMR and MS.

The solid is dissolved in pyridine and cooled to 0° C. A solution ofpyridine-HF complex was then added to the solution, which is stirred for8 hrs after the addition is complete. The reaction mixture is thenslowly added to a sat. sodium bicarbonate solution at 0° C. and the pHof the aqueous layer maintained above 7.0. When addition is complete,the aqueous solution is extracted with ethyl acetate (2×), and theorganic layer is washed with water and dried. Concentration affords asolid which is immediately dissolved in methanol, and sodium methoxidein methanol is added until the pH of the solution is above 14. Thereaction mixture is stirred at 40° C. while maintaining the pH of thereaction mixture above pH 12. When the reaction is complete, thesolution is neutralized with acetic acid and the solution concentratedto dryness. Chromatography (silica) is performed on the residue and theappropriate fractions are collected, combined and concentrated. Thestructure of the product is verified by NMR and MS.

2.4 Preparation of Tetra-Antennary-Glycan-Oxazaline (FIG. 2B.2)

The synthesized tetra-antennary-N-linked glycan (0.5 g) is added to asolution containing pyridine (20 mL) and DMAP (0.1 g). The solution iscooled to 0° C., and acetic anhydride (400 mole eq) is slowly added. Thereaction is warmed to 40° C. until the reaction is complete asdetermined by TLC. The reaction mixture is concentrated to dryness andethyl acetate is added to dissolve the residue. The organic layer iswashed with water, sat. sodium bicarbonate/water, water and is thendried (Na₂SO₄). After filtration, the filtrate is concentrated todryness and chromatography (silica) performed on the residue.Appropriate fractions are collected, concentrated and characterized byNMR and MS.

The resulting solid is dissolved in dichloromethane and BF₃ added andthe reaction mixture is stirred at room temperature. When the reactionis complete by TLC, the reaction mixture is washed with water/sodiumbicarbonate and dried. The mixture is then filtered and the filtrate isconcentrated.

The residue is then dissolved in methanol, and sodium methoxide inmethanol is added until the pH of the solution is above 14. The reactionmixture is stirred at 40° C., while maintaining the pH of the reactionmixture above pH 12. When the reaction is complete, the solution isneutralized with acetic acid and concentrated to dryness. Chromatography(silica) is then performed on the residue and appropriate fractions arecollected. These are combined, concentrated and the structure verifiedby NMR and MS.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1. An in vitro method of glycosylating a polypeptide comprising an Asnor an Asp residue, the method comprising the steps of contacting thepolypeptide with a glycosyl donor molecule having a GlcNAc residue and aPNGase-F amidase under conditions suitable for the linkage of the GlcNAcresidue on the glycosyl donor molecule to the Asn or Asp residue on thepolypeptide, wherein said PNGase-F amidase comprises at least one aminoacid substitution of an amino acid residue for an active site acidicamino acid residue selected from the group consisting of Asp at position60, Glu at position 206 and Glu at position 118 corresponding to awild-type PNGase-F amidase sequence (SEQ ID NO:01).
 2. The method ofclaim 1, wherein the glycosyl donor molecule is modified with a leavinggroup at the reducing terminus of the molecule.
 3. The method of claim2, wherein the leaving group is a halogen.
 4. The method of claim 3,wherein the halogen is fluoride.
 5. The method of claim 2, wherein theleaving group is a Asn, or a Asn-peptide moiety.
 6. The method of claim1, wherein the GlcNAc residue on the glycosyl donor molecule ismodified.
 7. The method of claim 6, wherein the GlcNAc residue comprisesa 1,2 oxazoline moiety.
 8. The method of claim 1, wherein the glycosyldonor molecule comprises a bi, tri, or tetra-antennary structure.
 9. Themethod according to claim 1, wherein the glycosyl donor comprises alinkage between GlcNAc and mannose.
 10. The method according to claim 1,wherein the glycosyl donor comprises a high mannose N-linked structure.11. The method according to claim 1, wherein the glycosyl donorcomprises mannose-6-phosphate.
 12. The method of claim 1, wherein thePNGase-F amidase is attached to a solid support.
 13. The method of claim1, wherein the glycopeptide is reversibly attached to a solid support.14. The method of claim 1, further comprising the step of recombinantlyexpressing the polypeptide in a prokaryotic cell.
 15. The method ofclaim 13, wherein the prokaryotic cell is a bacterial cell.
 16. Themethod of claim 1, further comprising the step of recombinantlyexpressing the polypeptide in an eukaryotic cell.
 17. The method ofclaim 16, wherein the eukaryotic cell is a yeast cell or an insect cell.18. The method of claim 16, further comprising the step of contactingthe polypeptide with a wild type amidase to cleave carbohydratestructures from the polypeptide before the step of contacting thepolypeptide with the PNGase-F amidase.
 19. The method according to claim1, wherein the polypeptide comprises an acceptor moiety for aglycosyltransferase, and the method further comprises contacting thepolypeptide with a reaction mixture that comprises a glycosyl donormoiety and a glycosyltransferase under conditions appropriate totransfer a glycosyl residue from the glycosyl donor moiety to theglycosyltransferase acceptor moiety.
 20. A composition comprising aglycopeptide glycosylated according to the method of claim
 1. 21. Thecomposition of claim 20, wherein at least 80% of the acceptor moietieson the glycopeptide are glycosylated.
 22. The composition of claim 20,wherein glycopeptide is attached to a solid support.
 23. The compositionof claim 20, wherein the glycopeptide is a full-length glycopeptide. 24.The composition according to claim 20, wherein the glycopeptide is on acell.
 25. A large-scale in vitro method for modifying the glycosylationpattern of a polypeptide comprising an acceptor moiety for a PNGase-Famidase, the method comprising: contacting at least about 500 mg of thepolypeptide with a reaction mixture that comprises a glycosyl donormoiety for the PNGase-F amidase under conditions appropriate to transfera glycosyl residue from the glycosyl donor moiety to the acceptormoiety, thereby producing the glycopeptide having modified glycosylationpatterns, wherein said PNGase-F arnidase comprises at least one aminoacid substitution of an amino acid residue for an active site acidicamino acid residue selected from the group consisting of Asp at position60, Glu at position 206 and Glu at position 118 corresponding to awild-type PNGase-F amidase sequence (SEQ ID NO:01).
 26. The methodaccording to claim 25, wherein the modified glycosylation pattern is asubstantially uniform glycosylation pattern.
 27. The method according toclaim 25, wherein the polypeptide is a recombinant polypeptide.
 28. Themethod according to claim 25, wherein the polypeptide comprises anacceptor moiety for a glycosyltransferase, and the method furthercomprises contacting the polypeptide with a reaction mixture thatcomprises a glycosyl donor moiety and a glycosyltransferase underconditions appropriate to transfer a glycosyl residue from the glycosyldonor moiety to the glycosyltransferase acceptor moiety.
 29. A peptideprepared by a method according to claim
 25. 30. A large-scale in vitromethod of producing a glycopeptide, the method comprising: (a)contacting at least about 500 mg of a polypeptide with a reactionmixture that comprises a glycosyl donor moiety and a PNGase-F amidaseunder conditions appropriate to transfer a glycosyl residue from theglycosyl donor moiety to a glycosyl acceptor moiety on the polypeptide;and (b) terminating the transfer of the glycosyl residue to the glycosylacceptor when the glycosylation pattern is substantially identical tothe known glycosylation pattern is obtained, wherein said PNGase-Famidase comprises at least one amino acid substitution of an amino acidresidue for an active site acidic amino acid residue selected from thegroup consisting of Asp at position 60, Glu at position 206 and Glu atposition 118 corresponding to a wild-type PNGase-F amidase sequence (SEQID NO:01).
 31. The method according to claim 30, wherein the terminatingis due to exhausting in the reaction mixture a member selected from thePNGase-F amidase, the glycosyl donor, the glycosyl acceptor, quench witha chelator and combinations thereof.
 32. The method according to claim30, wherein the polypeptide is a recombinant polypeptide.
 33. A peptideprepared by a method according to claim 30.